Breath analysis system

ABSTRACT

A breath analysis system that includes a handle assembly with an analysis cartridge on an upper end thereof. The handle includes a main body portion with a pressure opening and a pressure transducer therein. The analysis cartridge includes a main body portion with an upper portion that defines a breath chamber, a lower portion that defines a fluid chamber and a filter assembly that is movable between a breath capture position and an analysis position. The filter assembly has an opening defined therethrough. In the breath capture position, the opening partially defines the breath chamber and in the analysis position the opening partially defines the fluid chamber. The system also includes an analysis device with a case, a door, a controller that controls the motor and a fluorescence detection assembly and a rotation assembly positioned in the case interior. The rotation assembly includes a shroud with a funnel portion for receiving the analysis cartridge.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/156,441, filed May 4, 2015, U.S. Provisional Application No.62/149,988, filed Apr. 20, 2015, and U.S. Provisional Application No.62/018,448, filed Jun. 27, 2014, which are all incorporated by referenceherein in their entireties.

FIELD OF THE INVENTION

The present invention is directed to the field of carbonyl detection andquantitation, and in particular the detection and quantitation of theconcentration of carbonyl containing moieties in biological samples.

BACKGROUND OF THE INVENTION

The detection of carbonyl containing moieties is known but the precisedetection of specific low concentrations of specific carbonyl containingmoieties in biological samples is not known. The use of carbonyl's toinduce the polymerization of o-phenylene diamine and p-phenylene diamineat high temperature is known to produce solid polymers for subsequentuse in manufacturing products, but the use of phenylene diaminederivatives is not known to be used in methods to detect carbonylcontaining moieties in a number of biological samples. In addition,measuring the fluorescence of a fluorogenic species in solution todetermine the presence of molecules corresponding to the species isknown, as well as the quantitation of the concentration of suchmolecules in a given sample. In addition, breath analysis deviceincluding for alcohol levels are known.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the breath analysis system in accordancewith a preferred embodiment of the present invention with the dooropened to show the analysis cartridge in the pocket;

FIG. 2 is a cross-sectional elevational view of the analysis cartridge;

FIG. 3A is a cross-sectional perspective view of the analysis cartridge;

FIG. 3B is a cross-sectional exploded perspective view of the analysiscartridge;

FIG. 4 is an exploded view of the handle assembly;

FIG. 5A is a cross-sectional perspective view of the analysis cartridgeprior to being connected to the handle assembly;

FIG. 5B is a cross-sectional perspective view of the analysis cartridgeafter being connected to the handle assembly;

FIG. 6A is a perspective view of the analysis cartridge prior to beingconnected to the handle assembly;

FIG. 6B is a perspective view of the analysis cartridge connected to thehandle assembly;

FIG. 7 is a bottom plan view of the analysis cartridge;

FIG. 8 is a perspective view of the back of the analysis device;

FIG. 9 is a perspective view of the back of the analysis device with thebattery cover removed;

FIG. 10 is a perspective view of the analysis device with half of thecase removed;

FIG. 11 is an exploded perspective view of the analysis device;

FIG. 12 is a top perspective view of the rotation assembly with theanalysis cartridge in the pocket;

FIG. 13 is an exploded perspective view of the rotation assembly;

FIG. 14 is another perspective view of bottom perspective view of therotation assembly;

FIG. 15 is a perspective view of the rotation assembly with the analysiscartridge in the pocket;

FIG. 16 is a perspective view of the rotatable portion with the analysiscartridge in the pocket;

FIG. 17A is an exploded perspective view of the rotatable portion withthe analysis cartridge in the pocket;

FIG. 17B is another exploded perspective view of the rotatable portion;

FIG. 17C is an exploded perspective view of the optical system;

FIG. 17D is a plan view of the bottom half of the optical systemhousing;

FIG. 17E is a perspective view of the optical system;

FIG. 18 is a perspective view of the second fixed member that includesthe cam track;

FIG. 19A is a perspective view of the rotation assembly with the arm inthe stowed position;

FIG. 19B is a perspective view of the rotation assembly with the arm inthe deployed position;

FIG. 20 is a perspective view of a portion of the rotatable assemblywith the second halve of the housing removed to show the components ofthe optical system;

FIG. 21A is a cross-sectional end view of the rotation assembly showingthe rotatable portion in the first position (also referred to as thestart position);

FIG. 21B is a cross-sectional end view of the rotation assembly showingthe rotatable portion in the second position (also referred to as thefirst mixing position);

FIG. 21C is a cross-sectional end view of the rotation assembly showingthe rotatable portion in the third position (also referred to herein asthe baseline reading position);

FIG. 21D is a cross-sectional end view of the rotation assembly showingthe arm in a stowed position and the rotatable portion rotating towardthe fourth position;

FIG. 21E is a cross-sectional end view of the rotation assembly showingthe rotatable portion in a fourth position (also referred to as theinsertion position) and the arm in the deployed position;

FIG. 22 is a cross-sectional end view of the rotation assembly showingthe rotatable portion in the fifth position (also referred to as theanalysis position);

FIG. 23 is a cross-sectional end view of the rotation assembly showingthe rotatable portion in the sixth position, where the analysiscartridge can be removed;

FIG. 24 is an exploded perspective view of an analysis cartridge systemthat includes a breath analysis cartridge and a fluorescence analysiscartridge in accordance with another preferred embodiment of the presentinvention;

FIG. 25 is a cross-sectional view of the breath analysis cartridge ofFIG. 24 with the ampule assembly in elevation;

FIG. 25A is a cross-sectional view of the ampule assembly of the breathanalysis cartridge;

FIG. 26 is a cross-sectional view of the breath analysis cartridge ofFIG. 24 with the ampule assembly in elevation and the ampule memberpushed in;

FIG. 26A is a cross-sectional view of the ampule assembly of the breathanalysis cartridge;

FIG. 27 is a cross-sectional view of the fluorescence analysis cartridgeof FIG. 24;

FIG. 28 is an elevational view of the analysis cartridge system of FIG.24 with the breath analysis cartridge received on the fluorescenceanalysis cartridge;

FIG. 29 is a cross-sectional view of the analysis cartridge system ofFIG. 24;

FIG. 30 is a cross-sectional view of an analysis cartridge in accordancewith another preferred embodiment of the present invention;

FIG. 31 shows graphs depicting the emission spectrum of the reaction ofmPDA with 1-hexanal as a function of time;

FIG. 32 shows a graph depicting the increase in fluorescence over timeof the reaction of mPDA with 1-hexanal being the carbonyl containingmoiety;

FIG. 33A shows a graph depicting the increase in fluorescence over timeof the reaction with 1-hexanal as a function of sodium dodecyl sulfate(“SDS”) concentration from 0.01 to 0.4% (w/v);

FIG. 33B shows a graph depicting the increase in fluorescence over timeof reaction with 1-hexanal as compared to a blank, with SDSconcentration at 0.2% SDS;

FIG. 33C shows a graph depicting the increase in fluorescence over timeof the reaction with 1-hexanal as compared to a blank, with SDSconcentration at 0.4% SDS;

FIG. 34 shows a graph displaying fluorescence as a function of 1-hexanalconcentration;

FIG. 35 shows a chart depicting the relative fluorescence as a functionof aldehyde chain length; and

FIG. 36 shows a chart depicting the relative fluorescence of selectedsmall aromatic amines.

SUMMARY OF THE PREFERRED EMBODIMENTS

In accordance with an aspect of the present invention there is providedan analysis cartridge that includes a main body portion having an upperportion that defines an upper chamber and a lower portion that defines afluid chamber, and a filter assembly that is movable along a filterassembly path between a first position and a second position. The filterassembly has an opening defined therethrough. In the first position, theopening partially defines the upper chamber and in the second positionthe opening partially defines the fluid chamber. In a preferredembodiment, the filter assembly is movable within a cylindrical sleevethat extends from the upper chamber to the fluid chamber. Preferably,the sleeve includes a top opening such that an upper surface of thefilter assembly is exposed to an exterior of the main body portion.

In a preferred embodiment, the filter assembly includes a cylindricallyshaped filter holder that includes the opening extending transverselytherethrough and two filters positioned such that they span the opening.The filters define a substrate space therebetween and a substrate isdisposed in the substrate space. In a preferred embodiment, thesubstrate is silica and the fluid chamber includes an elution solutionor rinse therein. Preferably, the upper chamber is a breath chamber thatincludes a breath entry opening, a breath exit opening and a breath paththerebetween. In a preferred embodiment, the analysis cartridge includesa pressure measurement hole defined in a wall of the upper portion thatcommunicates the breath chamber with a pressure tunnel that extendsthrough the main body portion and to a pressure recess defined in thelower portion.

In a preferred embodiment, a phenylene diamine derivative is disposed inthe analysis cartridge. Preferably, the analysis cartridge includes anampule member having a fluorescence chromophore space with the phenylenediamine derivative disposed therein and the fluid chamber includes anelution solution disposed therein. The ampule member is movable betweena first position where the phenylene diamine derivative is separatedfrom the elution solution and a second position where the phenylenediamine derivative is disposed in the elution solution. In a preferredembodiment, the phenylene diamine derivative is m-phenylene diamine.

In accordance with an embodiment of the present invention there isprovided a method that includes (a) obtaining an analysis cartridge thatincludes a main body portion having an upper portion that defines abreath chamber and a lower portion that defines a fluid chamber, and afilter assembly that is movable along a filter assembly path between afirst position and a second position. The filter assembly has an openingdefined therethrough and in the first position the opening partiallydefines the upper chamber and in the second position the openingpartially defines the fluid chamber and the fluid chamber includes anelution solution therein, as described below. The method also includes(b) capturing a breath sample in the filter assembly, (c) moving thefilter assembly from the first position to the second position, and (d)eluting constituents of the breath sample into the elution solution toform a constituent solution. In a preferred embodiment, prior to step(c) the method includes inserting the analysis cartridge into ananalysis pocket, and rotating the analysis cartridge to an insertionposition where an arm performs step (c).

In accordance with another embodiment of the present invention there isprovided an analysis cartridge system that includes a breath analysiscartridge and a fluorescence analysis cartridge. The breath analysiscartridge includes a main body portion that includes an upper portionthat defines a breath chamber and a lower portion that defines a fluidchamber. The breath chamber includes a breath entry opening, a breathexit opening and a breath path therebetween, and the lower portionincludes a receiver member extending therefrom. The breath analysiscartridge also includes a filter assembly that is movable along a filterassembly path between a first position and a second position. The filterassembly has an opening defined therethrough, and, in the firstposition, the opening partially defines the breath chamber and is partof the breath path and in the second position the opening partiallydefines the fluid chamber. The fluorescence analysis cartridge includesa main body portion that includes an upper portion that defines an upperchamber and a lower portion that defines a fluid chamber. The upperchamber includes a front opening that is adapted to receive the receivermember of the breath analysis cartridge therein. The fluorescenceanalysis cartridge also includes a filter assembly that is movable alonga filter assembly path between a first position and a second positionThe filter assembly has an opening defined therethrough, and, in thefirst position, the opening partially defines the upper chamber and inthe second position the opening partially defines the fluid chamber. Ina preferred embodiment, the in both the breath analysis cartridge andthe fluorescence analysis cartridge the breath or upper chamber issealed from the fluid chamber when the filter assembly is in the firstposition.

In a preferred embodiment, the breath analysis cartridge includes anampule member that is slideable within a slide tube between the firstand second positions. Preferably, the ampule member includes at leastone opening therein that is sealed from fluid communication with thefluid chamber when the ampule member is in the first position, and is influid communication with the fluid chamber when the ampule member is inthe second position. In a preferred embodiment, the filter assemblydivides the upper chamber of the fluorescence analysis cartridge into afront chamber and a rear chamber. The front chamber includes a piercingmember disposed therein that is adapted to pierce a breakable barrier ofthe ampule member. Preferably, the rear chamber of the fluorescenceanalysis cartridge includes an absorption member positioned therein.

In a preferred embodiment, the breath analysis cartridge includes aremovable mouthpiece that defines a central opening that is incommunication with the breath chamber. The mouthpiece includes a sleeveportion that is received in the breath entry opening and a mouthpieceportion. Preferably, the mouthpiece includes a stopper that abuts themain body portion. The stopper includes an alignment member extendingtherefrom that is received in an alignment opening in the main bodyportion. In a preferred embodiment, the fluorescence analysis cartridgeincludes opposing light entry and light exit windows positioned onopposite sides of the fluid chamber, and a fluorescence windowpositioned on the bottom of the main body portion. Preferably, the lightentry and light exit windows each include an outer surface, and whereinthe outer surfaces are parallel to one another. Preferably, thefluorescence window includes an outer surface and the outer surface ofthe fluorescence window is perpendicular to the outer surface of thelight entry window.

In accordance with another embodiment of the present invention there isprovided a method that includes obtaining an analysis cartridge systemthat includes a biological analysis cartridge and an identifiedconstituent analysis cartridge. The biological analysis cartridge has anupper chamber and a fluid chamber, and the identified constituentanalysis cartridge has an upper chamber and a fluid chamber. The methodalso includes capturing a biological sample as described below on asubstrate as described below positioned in the upper chamber in thebiological analysis cartridge, moving the substrate from the upperchamber to the fluid chamber, which includes a first elution solutiontherein as described below, eluting constituents of the biologicalsample into the first elution solution to form a constituent solution asdescribed below, releasing a moiety into the second solution to form afirst identifiable constituents solution, transferring the firstidentifiable constituents solution as described below to the upperchamber of the identified constituent analysis cartridge, such thatidentified constituents are captured on a substrate positioned in theupper chamber, moving the substrate from the upper chamber to a fluidchamber that includes a second elution solution therein as describedbelow, and eluting the identified constituents into the second elutionsolution to form a second identifiable constituents solution asdescribed below.

In a preferred embodiment, the biological analysis cartridge is a breathanalysis cartridge, the identified constituent analysis cartridge is afluorescence analysis cartridge, and the biological sample is a breathsample. Preferably, the moiety is a fluorescence chromophore asdescribed below.

In accordance with another embodiment of the present invention there isprovided a method of forming a solution within a breath analysiscartridge that includes a main body portion with an upper portion thatdefines a breath chamber a lower portion that defines a fluid chamberhaving an elution solution disposed therein and an ampule member that ismovable between a first position and a second position. The ampulemember includes a fluorescence chromophore space having a fluorescencechromophore disposed therein. The method includes moving the ampulemember from the first position where the fluorescence chromophore spaceand fluorescence chromophore are separated from the fluid chamber to thesecond position where the fluorescence chromophore space is incommunication with the fluid chamber, and mixing the fluorescencechromophore with the elution solution.

In accordance with another embodiment of the present invention there isprovided an analysis cartridge that includes a main body portion thatincludes an upper portion that defines a breath chamber and a lowerportion that defines a fluid chamber. The breath chamber includes abreath entry opening, a breath exit opening and a breath paththerebetween. The analysis cartridge also includes a filter assemblythat is movable along a filter assembly path between a first positionand a second position. The filter assembly has an opening definedtherethrough, and, in the first position, the opening partially definesthe breath chamber and is part of the breath path and in the secondposition the opening partially defines the fluid chamber. In a preferredembodiment, the filter assembly includes first and second filterspositioned in the opening and the first and second filters define asubstrate space therebetween with a substrate disposed therein.Preferably, the substrate is incorporated with an active reactivecapture agent. In a preferred embodiment, the active reactive captureagent is a fluorescent hydrazine or aminooxy compound.

In accordance with another embodiment of the present invention there isprovided a method of forming a fluorescing solution within an analysiscartridge that includes a main body portion with an upper portion thatdefines a breath chamber, a lower portion that defines a fluid chamberhaving an elution solution disposed therein and a filter assembly thatis movable along a filter assembly path between a first position and asecond position. The filter assembly has an opening definedtherethrough, and, in the first position, the opening partially definesthe breath chamber and in the second position the opening partiallydefines the fluid chamber. The filter assembly includes a substrateincorporated with an active reactive capture agent disposed therein. Themethod includes capturing carbonyl containing moieties on the substrate,moving the filter assembly from the first position to the secondposition, and eluting the carbonyl containing fluorescence chromophoresand active reactive capture agent into the elution solution to form thefluorescing solution.

In accordance with another embodiment of the present invention there isprovided a breath capture assembly that includes a handle assemblyhaving an elongated main body portion that defines a handle interior, acap disposed at an end of the main body portion that includes a pressureopening defined therein, and a pressure transducer disposed in thehandle interior. The breath capture assembly also includes an analysiscartridge received on an upper end of the handle assembly. The analysiscartridge includes a main body portion that has an upper portion thatdefines a breath chamber and a lower portion that defines a fluidchamber. The breath chamber includes a breath entry opening, a breathexit opening and a breath path therebetween. The analysis cartridgeincludes a filter assembly that is movable along a filter assembly pathbetween a first position and a second position. The filter assembly hasan opening defined therethrough, and, in the first position, the openingpartially defines the breath chamber and is part of the breath path andin the second position the opening partially defines the fluid chamber.

In a preferred embodiment, the pressure measurement hole is defined in awall of the upper portion of the analysis cartridge and communicates thebreath chamber with a pressure tunnel that extends through the main bodyportion. A pressure path is defined from the breath chamber, through thepressure measurement hole, the pressure tunnel, the pressure opening andto the pressure transducer. Preferably, the cap of the handle assemblyincludes a pressure protrusion extending upwardly therefrom that issealingly received in a pressure recess in the analysis cartridge. Thepressure recess is in communication with the pressure tunnel, and thepressure opening is defined in the pressure protrusion. In a preferredembodiment, the cap includes a seat defined therearound, and a collardepending downwardly from the analysis cartridge is received on theseat. The cap preferably includes an attachment protrusion extendingradially outwardly therefrom that is received in an attachment recessdefined in the collar of the analysis cartridge.

In a preferred embodiment, a hollow extension tends downwardly from thecap of the handle assembly and into the handle interior. The hollowextension is part of the pressure path. Preferably, a pressure tube isreceived on the hollow extension and is in the pressure path between thehollow extension and the pressure transducer.

In a preferred embodiment, the analysis cartridge includes a breakablebarrier disposed between the breath chamber and the fluid chamber whenthe filter assembly is in the first position to seal the breath chamberfrom the fluid chamber.

In accordance with another embodiment of the present invention there isprovided an analysis device that includes a case defining a caseinterior, a door movable between an open and a closed position, and arotation assembly positioned in the case interior that includes firstand second fixed members and a rotatable portion positioned between thefirst and second fixed members. The rotatable portion is rotatable abouta rotation axis with respect to the first and second fixed members. Therotatable portion includes a shroud that has a funnel portion definedtherein for receiving an object to be rotated. The shroud includes apocket opening defined at the top thereof and the rotation assemblyincludes a fluorescence detection assembly positioned generally belowthe shroud. The analysis device also includes a motor that drivesrotation of the rotatable portion and a controller that controls themotor and the fluorescence detection assembly.

In a preferred embodiment, the shroud includes a pocket opening and ananalysis opening opposite to one another, and the shroud includes wallsthat taper between the pocket opening and the analysis opening. Thefluorescence detection assembly preferably includes a housing that hasan analysis cartridge receiving portion with a well defined therein thatis aligned with the analysis opening in the shroud to form an analysispocket. In a preferred embodiment, the analysis cartridge receivingportion cooperates with the shroud to define a light entry aperture, alight exit aperture and a fluorescence aperture. Preferably, thefluorescence detection assembly includes a light that is configured tobe directed along a light path that extends through a light chamberdefined in the housing, through the light entry aperture, through thelight exit aperture, and into a light trap.

In a preferred embodiment, the fluorescence detection assembly includesa detector for receiving fluorescence emitted through the fluorescenceopening and through a fluorescence chamber defined in the housing.Preferably, the fluorescence chamber is generally orthogonal to thelight chamber. In a preferred embodiment, the analysis device includesan arm that is pivotal between a stowed position and a deployedposition. The arm includes a first end that extends through an armopening defined in the shroud when in the deployed position. When therotatable portion rotates from a start position to an insertion positionthe arm pivots from the stowed position to the deployed position. In apreferred embodiment, the arm is biased toward the stowed position andincludes a second end that is operationally associated with a camsurface on the second fixed member. The cam surface preferably has astowed end that is associated with the stowed position of the arm and adeployed end that is associated with the deployed end of the arm andincludes an increasing radius from the stowed end to the deployed end.In a preferred embodiment, the arm includes a ball bearing on the secondend thereof that interacts with the cam surface. Preferably, the arm ispivotal on a shaft that extends from the shroud.

In accordance with another embodiment of the present invention there isprovided a rotation assembly that includes first and second fixedmembers, and a rotatable portion positioned between the first and secondfixed members that is rotatable about a rotation axis with respect tothe first and second fixed members. The rotatable portion includes ashroud that has a funnel portion defined therein for receiving an objectto be rotated and an arm that is pivotal between a stowed position and adeployed position. The arm includes a first end that extends through anarm opening defined in the shroud when in the deployed position. Theanalysis device also includes a motor that drives rotation of therotatable portion. When the rotatable portion is rotated from a startposition to an insertion position the arm pivots from the stowedposition to the deployed position. In a preferred embodiment, the shroudincludes a pocket opening and an analysis opening opposite to oneanother and walls that taper between the pocket opening and the analysisopening.

In a preferred embodiment, the shroud includes first and second axlemembers extending outwardly therefrom that are received in opening inthe first and second fixed members, respectively. Preferably, the shroudincludes at least one internally threaded fastener receiver memberextending therefrom. The housing of the fluorescence detection assemblyincludes at least one receiver tube and a threaded receiver extendsthrough the receiver tube and into the fastener receiver member tosecure the shroud to the housing. Preferably, the housing includes firstand second housing halves. A first receiver tube is located on the firsthousing half and a second receiver tube is located on the second housinghalf. The threaded receiver extends through the first and secondreceiver tubes and into the fastener receiver member to secure theshroud to the housing.

In accordance with another embodiment of the present invention there isprovided a handle assembly for use with a breath analysis system thatincludes an analysis cartridge and an analysis device. T handle includesan elongated main body portion that defines a handle interior, a capdisposed at an end of the main body portion that includes a pressureopening defined therein, a pressure transducer disposed in the handleinterior, and a pressure path defined between the pressure opening andthe pressure transducer. In a preferred embodiment, the cap includes apressure protrusion extending upwardly therefrom and the pressureopening is defined in the pressure protrusion. Preferably, the handleinterior includes a magnet disposed therein that interacts with a magnetin the analysis device. The magnet is positioned in a magnet recessdefined in the cap.

In accordance with another embodiment of the present invention there isprovided a filter assembly that includes a main body portion having agenerally cylindrical shape that defines a first axis, an openingdefined transversely through the main body portion that extendsgenerally perpendicularly to the first axis, first and second filtersspanning the opening and defining a substrate space therebetween, and asubstrate disposed in the substrate space. Preferably, the first andsecond filters comprise a plastic having pores defined therethrough. Ina preferred embodiment, the main body portion includes guide rails on anoutside surface thereof that extend generally parallel to the axis.Preferably, the main body portion includes a lower surface that includesat least one piercer extending downwardly therefrom.

In accordance with another embodiment of the present invention there isprovided a method of making a filter assembly that includes obtaining afilter holder having a main body portion with a generally cylindricalshape that defines a first axis and includes an opening definedtransversely through the main body portion that extends generallyperpendicularly to the first axis, dosing a first filter with asubstrate, pressing a second filter onto the substrate, and positioningthe first filter, substrate and second filter into the opening such thatthe first and second filter span the opening. The first and secondfilters and substrate can be positioned in the opening together orseparately.

In accordance with another embodiment of the present invention there isprovided a fluorescence detection assembly that includes an emitter, adetector, a housing that defines an light chamber, a fluorescencechamber and a well, a light path that extends from the emitter, throughthe light chamber and through the well, and a fluorescence path thatextends from the well, through the fluorescence chamber and to thedetector. In a preferred embodiment, the fluorescence detection assemblyincludes a first lens and a first filter positioned within the lightpath. Preferably, the fluorescence detection assembly includes secondlens and a second filter positioned within the fluorescence path. In apreferred embodiment, the fluorescence detection assembly includes atleast one of a first light baffle positioned within the light pathbetween the emitter and the first lens, a second light baffle positionedwithin the light path between the first lens and the first filter and athird light baffle positioned within the light path between the firstfilter and the well. The first baffle includes a first light baffleaperture defined therein that has a smaller inner diameter than an innerdiameter of the light chamber. The second baffle includes a second lightbaffle aperture defined therein that has a smaller inner diameter thanthe inner diameter of the first light baffle aperture. The third baffleincludes a third light baffle aperture defined therein that has asmaller inner diameter than the inner diameter of the second lightbaffle aperture.

In a preferred embodiment, the light trap is positioned at a distal endof the light path and includes a first wall that is angled between about25° and about 45° with respect to the light path. Preferably, the lighttrap includes a second wall connected to the first wall and the secondwall is not perpendicular to the light path. In a preferred embodiment,the housing is comprised of an upper housing half and a lower housinghalf and the lower housing half includes an analysis cartridge receivingportion that defines the well. In a preferred embodiment, the upperhousing half includes a flange that extends downwardly therefrom andoverlaps a flange extending upwardly from the lower housing half. In apreferred embodiment, an analysis cartridge is positioned in the wellthat includes a light entry window, a light exit window and afluorescence window. The light entry window and light exit window arepositioned along the light path.

In a preferred embodiment, the housing is comprised of an upper housinghalf and a lower housing half that cooperate to define a first lenspocket that houses the first lens, a first filter pocket that houses thefirst filter, a second lens pocket that houses the second lens, and asecond filter pocket that houses the second filter. Preferably, thefluorescence detection assembly includes a shroud connected to thehousing that includes a pocket opening and an analysis opening oppositeto one another and a funnel portion therebetween. The funnel portioncooperates with the well to define an analysis pocket and the shroud atleast partially defines the light entry aperture and the fluorescenceaperture.

In accordance with another embodiment of the present invention there isprovided a method of detecting fluorescence that includes emitting lightfrom an emitter into an light chamber and along a light path thatincludes a sensing chamber therealong. The sensing chamber includes afluorescing solution therein. The emitted light passes through thefluorescence solution and produces a fluorescence light, and wherein thefluorescence light is emitted from the sensing chamber into afluorescence chamber along a fluorescence path, and detecting afluorescence signal of the fluorescence light.

In accordance with another embodiment of the present invention there isprovided a method of detecting fluorescence that includes inserting ananalysis cartridge into an analysis pocket. The analysis cartridgeincludes a filter assembly that includes a substrate having a carbonylcontaining moiety thereon. The method also includes rotating theanalysis cartridge from a start position to an insertion position,moving the filter assembly within the analysis cartridge from an upperchamber to a fluid chamber that contains an elution solution, rotatingthe analysis cartridge from the insertion position to an analysisposition such that the elution solution drains through the filterassembly and the carbonyl containing moiety is eluted into the elutionsolution to form a fluorescing solution, and analyzing the fluorescenceof the fluorescing solution.

In accordance with another embodiment of the present invention there isprovided a breath analysis system that includes a breath captureassembly that includes a handle assembly that includes an elongated mainbody portion that defines a handle interior, a pressure opening definedin an end of the elongated main body portion, and a pressure transducerdisposed in the handle interior. The breath analysis system alsoincludes an analysis cartridge received on an upper end of the handleassembly. The analysis cartridge includes a main body portion thatincludes an upper portion that defines a breath chamber, and a lowerportion that defines a fluid chamber. The breath chamber includes abreath entry opening, a breath exit opening and a breath paththerebetween. The analysis cartridge includes a filter assembly that ismovable along a filter assembly path between a breath capture positionand an analysis position. The filter assembly has an opening definedtherethrough, and, in the breath capture position, the opening partiallydefines the breath chamber and is part of the breath path and in theanalysis position the opening partially defines the fluid chamber. Thesystem also includes an analysis device that includes a case defining acase interior, a door movable between an open and closed position, and arotation assembly positioned in the case interior that includes a shroudthat has a funnel portion defined therein for receiving the analysiscartridge. The system also includes a controller that controls the motorand the fluorescence detection assembly. The pressure transducer is incommunication with the controller.

In accordance with another embodiment of the present invention there isprovided a method for detecting and quantifying carbonyl containingmoieties in breath. The method includes (a) providing an analysiscartridge, (b) connecting the analysis cartridge to a handle assembly,(c) collecting a breath sample of carbonyl containing moieties on afilter assembly, (d) labeling the carbonyl containing moieties toprovide a labeled solution, (e) inserting the labeled solution into ananalysis device, (f) directing light within a predetermined wavelengthrange through the labeled solution, thereby producing a fluorescence,and (g) detecting the fluorescence.

It will be appreciated that any biological sample can be analyzed usingthe system. Breath constituents other than carbonyl containing moieties(CCM) or aldehydes can be captured and analyzed as desired. U.S. PatentPublication Nos. 2003/0208133 and 2011/0003395 are incorporated byreference herein in their entireties.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description and drawings are illustrative and are not tobe construed as limiting. Numerous specific details are described toprovide a thorough understanding of the disclosure. However, in certaininstances, well-known or conventional details are not described in orderto avoid obscuring the description. References to one or anotherembodiment in the present disclosure can be, but not necessarily are,references to the same embodiment; and, such references mean at leastone of the embodiments.

Reference in this specification to “one embodiment” or “an embodiment”means that a particular feature, structure, or characteristic describedin connection with the embodiment is included in at least one embodimentof the disclosure. Appearances of the phrase “in one embodiment” invarious places in the specification do not necessarily refer to the sameembodiment, nor are separate or alternative embodiments mutuallyexclusive of other embodiments. Moreover, various features are describedwhich may be exhibited by some embodiments and not by others. Similarly,various requirements are described which may be requirements for someembodiments but not other embodiments.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the disclosure, and in thespecific context where each term is used. Certain terms that are used todescribe the disclosure are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the disclosure. For convenience, certainterms may be highlighted, for example using italics and/or quotationmarks: The use of highlighting has no influence on the scope and meaningof a term; the scope and meaning of a term is the same, in the samecontext, whether or not it is highlighted. It will be appreciated thatthe same thing can be said in more than one way.

Consequently, alternative language and synonyms may be used for any oneor more of the terms discussed herein. Nor is any special significanceto be placed upon whether or not a term is elaborated or discussedherein. Synonyms for certain terms are provided. A recital of one ormore synonyms does not exclude the use of other synonyms. The use ofexamples anywhere in this specification including examples of any termsdiscussed herein is illustrative only, and is not intended to furtherlimit the scope and meaning of the disclosure or of any exemplifiedterm. Likewise, the disclosure is not limited to various embodimentsgiven in this specification.

Without intent to further limit the scope of the disclosure, examples ofinstruments, apparatus, methods and their related results according tothe embodiments of the present disclosure are given below. Note thattitles or subtitles may be used in the examples for convenience of areader, which in no way should limit the scope of the disclosure. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this disclosure pertains. In the case of conflict, thepresent document, including definitions, will control.

It will be appreciated that terms such as “front,” “back,” “top,”“bottom,” “side,” “short,” “long,” “up,” “down,” and “below” used hereinare merely for ease of description and refer to the orientation of thecomponents as shown in the figures. It should be understood that anyorientation of the components described herein is within the scope ofthe present invention.

FIGS. 1-30 show a breath analysis system 10 for analyzing carbonylcontaining moieties (“CCM”) in a patient's breath. As shown in FIG. 1,the system 10 generally includes a handle assembly 12, an analysiscartridge 14 and an analysis device 16. Generally, the handle assembly12 and analysis cartridge 14 are used by the clinician and patient tocapture certain components of the patient's breath (as described morefully below), and the analysis device 16 is used to analyze the capturedcomponents.

The analysis cartridge 14 shown in FIGS. 2-7 and 21A-23 will now bedescribed. In a preferred embodiment, the analysis cartridge 14 includesa main body portion 11 that includes an upper portion 29 that defines anupper or breath chamber 30 and a lower portion 31 that defines a loweror fluid chamber 32. The breath chamber 30 includes a front opening orbreath entry opening 33, a breath exit opening 40 and a breath path P1therebetween. In a preferred embodiment, the breath chamber 30 taperstoward the breath exit opening 40, however, this is not a limitation.The analysis cartridge 14 also includes a filter assembly 19 that ismovable along a filter assembly path P2 between a first or breathcapture position (FIG. 2) and a second or analysis position (FIG. 21E).The filter assembly 19 has an opening 17 defined therethrough thatincludes at least one and preferably two filters 26 positioned therein.In the breath capture position the opening 17 partially defines thebreath chamber 30 and is part of the breath path P1 and in the analysisposition the opening 17 partially defines the fluid chamber 32.

In a preferred embodiment, the analysis cartridge 14 includes aremovable mouthpiece 18, the filter assembly 19 on the top and an ampulemember 22 on the bottom. As shown in FIG. 3B, the mouthpiece 18 includesa sleeve portion 15 that is received in breath entry opening 33 on themain body portion 11, a mouthpiece portion 18 a, a stopper 21 that abutsthe main body portion 11 and an alignment member 21 a that is receivedin a complementary alignment opening in the main body portion 11 (notshown). The mouthpiece 18 partially defines the breath chamber 30 andthe breath path P1. The filter assembly 19 preferably includes twofilters or frit plates 26 (sometimes referred to together as a fritstack) that are held by a frit holder 20. The frit plates 26 spanopening 17.

In a preferred embodiment, the frit holder 20 includes at least onepiercer 20 a on the bottom surface thereof for piercing a breakablebarrier discussed below. Preferably, the frit holder 20 includes atleast one guide rail 39 on an outside surface for helping guide the fritassembly 19 as it is moved along the filter assembly path. The piercer20 a can be on the bottom of the guide rail 39. Prior to use, the fritstack 26 is positioned in the breath chamber 30. As shown in FIG. 2, asubstrate space 27 is defined between the frit plates 26. In a preferredembodiment, a substrate 28, such as silica, is disposed in the substratespace 27 between the frit plates 26. It will be appreciated that thefrit plates 26 are sufficiently porous so that the breath can passtherethrough, but not so porous that the substrate 28 trappedtherebetween can escape. The filters or frit plates 26 are preferablymade of polyethylene spheres that are pressed and packed together in aform. When pressed together in the disc or plate shape, the spherical orroundish shape creates the voids or pores necessary for breath to getthrough. The spheres can be made out of different plastic materials(e.g., polyethylene, polypropylene, etc.) or teflon in differentdiameters. In another embodiment, the filters 26 can comprise spheresall made of the same plastic materials and of the same or differentdiameters. In an exemplary embodiment, the frits 26 are polyethylene orteflon frits with 10 μm or 20 μm pore sizes. As is described more fullybelow, in use, as a patient blows through the breath chamber 30, CCMincluding aldehydes, collect on the substrate 28 (also referred to asCCM capture material). In a preferred embodiment, silica is used as thesubstrate or CCM capture material. However, this is not a limitation onthe present invention and other substrates with the ability to captureCCM or aldehydes can be used.

In a preferred embodiment, the frit plates or filters 26 that spanopening 17 are preferably press fit therein. The method of creating thefilter assembly includes pressing the spherical plastic pieces intofirst and second filters 26, pressing the first filter 26 into theopening 17 in the frit holder 20. Then, the substrate 28 (preferablysilica) is dosed on the first frit 26, a second filter 26 is thenpressed into the opening 17 onto the silica 28 using a predefinedpressure. In another embodiment, the silica 28 can be dosed onto thefirst frit 26 and then the second frit 26 can be pressed onto the silica28 to create a frit stack, prior to pressing the frit stack into theopening 17 in the frit holder 20. In another embodiment, the filters canbe disposed in grooves defined in the inside wall of the frit holder 20.

As shown in FIGS. 2-3B, the ampule member 22 comprises a main bodyportion 23 having a fluorescence chromophore space or trough 25 definedtherein that includes an upper rim 23 a and a lower surface 23 b. Thetrough 25 includes a phenylene diamine derivative (“PD derivative”) 24disposed therein. The ampule member 22 is movable between a firstposition where the trough 25 and PD derivative 24 are separated from thefluid chamber 32 by a first breakable barrier 36 a and a second positionwhere the trough 25 is in communication with the fluid chamber 32 (wherethe PD derivative 24 and elution solution 34 are mixed in the fluidchamber 32, as described below). The breakable barrier 36 a can be afoil or the like. In a preferred embodiment, the ampule member 22 ismovable within an ampule tunnel 130 that is defined in the lower portion31 of the analysis cartridge 14. In a preferred embodiment, the ampulemember 22 includes a flange or stopper 23 c that abuts a stopper surface132 on the analysis cartridge 14 when the ampule member 22 is moved tothe second position. The stopper 23 c prevents the ampule member 22 frommoving too far into the ampule tunnel 130 and/or into the fluid chamber32. In a preferred embodiment the ampule tunnel 130 is orthogonal to thefluid chamber 32. However, this is not a limitation.

As shown in FIGS. 2-3B, the fluid chamber 32 is located between thebreath chamber 30 and the ampule member 22 and ampule tunnel 130. Anelution solution 34 is disposed in the fluid chamber 32. In a preferredembodiment, the elution solution 34 includes water and ethanol, however,this is not a limitation on the present invention. In a preferredembodiment, the fluid chamber 32 is sealed from the ampule tunnel 130.This can be done by any sealing method. In a preferred embodiment, thefluid chamber 32 is sealed from the ampule tunnel 130 by first breakablebarrier 36 a. In a preferred embodiment, the fluid chamber 32 is sealedfrom the breath chamber 30. This can be done by any sealing method. In apreferred embodiment, a second breakable barrier 36 b positioned acrossthe filter assembly pathway P2 (dividing the filter assembly sleeve 53)between the breath chamber 30 and the fluid chamber 32. The openingbetween the fluid and breath chambers is referred to herein as thefilter assembly opening 134 and it includes a ledge on which the secondbreakable barrier 36 b is secured. The fluid chamber 32 also includesvent holes 37 to keep the elution solution 34 from getting “air locked”during mixing.

It will be appreciated by those of skill in the art that before use ofthe analysis cartridge 14 (i.e., before it is attached or connected tothe handle assembly 12), the filter assembly 19 is in the breath captureposition and the ampule member 22 is in the first position. In thisconfiguration, the elution solution 34 in the fluid chamber 32 isseparated from the filter assembly 19 in the breath chamber 30 by secondbreakable barrier 36 b and the ampule member 22 in the ampule tunnel 130by first breakable barrier 36 a.

FIG. 4 is an exploded view of the handle assembly 12 and the componentsthereof. In a preferred embodiment, the handle assembly 12 includes anelongated main body portion 101 with first and second halves 102 thatdefine a handle interior 99 (see FIG. 5A), top and bottom end caps 103and 105, a grip 104, a cable 106 that connects to the analysis device 16(electric and/or data) via plug 107 and a pressure transducer 50 andassociated components (circuit board 108, pressure tube 110, etc.). Thehandle assembly 12 also preferably includes a magnet 111 that interactswith a magnet 150 a in handle storage pocket 66 and tube 150 describedbelow. A pressure protrusion 49 extends outwardly from the upper surface52 of the top end cap 103 (see FIG. 5A). A pressure opening 113 isdefined in the top of the pressure protrusion 49. In a preferredembodiment, the pressure protrusion includes an O-ring 115 therearoundthat seals the pressure protrusion 49 when it is coupled to the analysiscartridge 14. Preferably, the pressure protrusion 49 is received in apressure recess 139 (see FIG. 7) defined in the lower portion 31 of theanalysis cartridge. As shown in FIGS. 5 and 5A, in a preferredembodiment, the pressure path within the handle assembly 12 extends fromthe pressure opening 113, through an extension 117 on the bottom surfaceof the top end cap 103 (which is received in the pressure tube 110),through the pressure tube 110 and to the pressure transducer 50 (an endof which is received in the pressure tube 110). Generally, the pressurepath is defined between the pressure opening 113 and the pressuretransducer 50. The cable 106 is connected to the circuit board 108.Therefore, the pressure reading of the pressure transducer 50 can becommunicated to main circuit board 74 of the analysis device 16.

FIGS. 5A-6B show the analysis cartridge 14 being attached to the handleassembly 12. In a preferred embodiment, the analysis cartridge 14includes a collar 138 extending downwardly from the main body portion11. The collar 138 includes at least one and preferably a plurality ofattachment recesses 140 defined therein. One of the recesses 140 mateswith an alignment or attachment protrusion 142 on the top end cap 103 ofthe handle assembly 12 (in another embodiment there can be moreattachment protrusions 142). The collar 138 is also received on a seat144 that is a part of the top end cap 103. An annular protrusion extendsoutwardly from cap 103 that mates with an undercut slot on the collar138 and that creates a snap fit. A friction fit is also within the scopeof the present invention. The attachment recesses 140 allow the collarto expand when secured on the top of the handle assembly 12.

The pressure recess 139 for receiving the pressure protrusion 49 on thetop of the handle assembly 12 is defined within the collar 138. Thecomplementary attachment recess 140 and attachment protrusion 142 alignthe analysis cartridge 14 and handle assembly 12 during the attachmentprocess so that the pressure protrusion 49 is received in the pressurerecess 139. The seat 144 can include a rubber material or the like forproviding a friction fit with the collar 138. Any method of attachingthe analysis cartridge 14 to the handle assembly 12 is within the scopeof the present invention. For example, analysis cartridge 14 to thehandle assembly 12 can be connected by a threaded fit, snap fit,friction fit or the like.

As shown in FIG. 5A, in the first position, the ampule member 22 extendsdownwardly from the lower portion 31 of the analysis cartridge 14.Therefore, when the analysis cartridge 14 is connected to the handleassembly 12, the lower surface 23 b of the ampule member 22 contacts theupper surface 52 of the handle assembly 12, thereby pushing the ampulemember 22 upwardly, thus breaking the first breakable barrier 36 a, andmoving the ampule member from the first position to the second position.This facilitates transferring the PD derivative 24 into the fluidchamber 32 and the elution solution 34. It will be appreciated that thePD derivative 24 is kept separated from the elution solution 34 untilthe analysis cartridge 14 is connected to the handle assembly 12. Theelution solution 34 and PD derivative 24 mix to form a phenylene diaminesolution (“PD solution”) 35 (further mixing of the elution solution 34and PD derivative 24 analysis device 16 is described below).

With references to FIGS. 2-7, another feature included in the analysiscartridge 14 is the breath pressure measurement capability. This enablesa patient blowing into the analysis cartridge to know via the screen 60on the analysis device 16 whether the blown pressure is within apredetermined range. Generally, a pressure path is defined between apressure measurement hole 42 in the upper portion 29 of the analysiscartridge 14 (that is in communication with the breath chamber 30) andthe pressure transducer 50 in the handle assembly 12. As shown in FIG.3A, the pressure path extends from the pressure measurement hole 42 to apressure channel 44 that extends partially around the filter assembly19, and to a pressure tunnel 46 that extends downwardly through the mainbody portion 11. It will be appreciated that FIG. 3A shows a top ringcover 47 omitted from the analysis cartridge 14. The top ring cover 47(or other wall or barrier) encloses the pressure channel 44 to maintainthe pressure.

FIG. 7 shows the bottom of the analysis cartridge 14 and the end of thepressure tunnel 46 that communicates with the pressure recess 139. Whenthe pressure protrusion 49 is received in the pressure recess 139, thepressure tunnel 46 is communicated with the pressure opening 113.Therefore, the complete pressure path extends from the pressuremeasurement hole 42, through the pressure channel, through the pressuretunnel, through the pressure opening, through the hollow extension 117,through the pressure tube 110 and to the pressure transducer 50.

When a patient blows through the breath chamber 30 (and the frit plates26), a pressure measurement is taken. In a preferred embodiment, thisrequires a pressure differential flow calculation. As breath is beingblown through the breath chamber, depending on how hard the person isblowing, there is a pressure differential that is created in the distalspace 38 of the breath chamber 30 in between the rear frit plate 26 andthe breath exit hole 40. The pressure measurement hole 42 is defined inthe wall within the distal space 38 and is essentially a tap formeasuring ambient and distal pressure differential. The pressure of thebreath in distal space 38 via pressure measurement hole 42 pressurizesthe existing air within the pressure channel (pressure path). Thepressure path extends through the pressure measurement hole 42 into thechannel 44 and is channeled over and then down through pressure tunnel46 and to the handle assembly 12. The pressure that is inside the distalspace 38 of the breath chamber 30 is being measured by pressuretransducer 50 and based on the pressure measurement the flow ratethrough the breath chamber 30 can be calculated. The electronics of thepressure transducer 50 are located in the handle assembly 12 (i.e., onthe circuit board 108).

In use, once the analysis cartridge 14, is placed on the handle assembly12, a user blows through the mouthpiece 18 and the breath chamber 30 sothat a predetermined volume of air or breath (e.g., 3 liters) is exhaledthrough the breath chamber 30. Therefore, CCM are filtered out of apredetermined or known volume of breath and are collected on thesubstrate 28. After the CCM have been collected, the user removes theanalysis cartridge 14 from the handle assembly 12, removes themouthpiece 18 and places the analysis cartridge 14 in the analysisdevice 16, as described below. The analysis cartridge 14 and handleassembly 12 in combination (shown in FIG. 6B) are referred to herein asthe breath capture assembly 13.

As shown in FIGS. 6A-7, the analysis cartridge includes a bottom windowand two side windows. The bottom window is referred to herein as thefluorescence window 170 and the side windows are referred to herein asthe light entry window 172 a and the light exit window 172 b. Thefluorescence window 170, light entry window 172 a and light exit window172 b are used in conjunction with an optical system 77 (also referredto herein as a fluorescence detection assembly or fluorometer) in theanalysis device 16 described below. In a preferred embodiment, thewindows 170 and 172 a and 172 b are a clear plastic and are the samematerial as the remainder of the main body portion 11. However, thewindows can be a different material. Preferably, the windows 170 and 172a and 172 b are optically polished and are oriented such that outersurface is orthogonal to the appropriate components of the opticalsystem 77 (described below). In a preferred embodiment, the remainder ofthe main body portion 11 is not optically clear and includes a draft sothat the windows 170 and 172 a and 172 b are isolated (i.e., the sidesor bottom of the cartridge are angled or not parallel to the outersurface of the windows). Preferably, the light entry and light exitwindows 172 a and 172 b are parallel to one another and the fluorescencewindow 170 is perpendicular to the light entry and light exit windows172 a and 172 b.

In a preferred embodiment, the analysis cartridge 14 is made of plastic(e.g., polycarbonate, PMMA, etc.) and the various pieces areultrasonically bonded to one another. However, this is not a limitationand the analysis cartridge can be made of any desirable material andbonded as desired.

FIGS. 1 and 8-23 show the analysis device 16. As shown in FIGS. 1 and8-11, generally, the analysis device 16 includes a case 48 (comprised oftwo halves 48 a and 48 b), a door 54 that is slidable between open andclosed positions, a shroud 56, analysis pocket 58 defined in the shroud56 (where the analysis cartridge is received), a bottom 57, a maincircuit board 74, a rotation assembly 76 and a display 60 (which ispreferably touch screen). It will be appreciated that the full analysispocket 58 includes the tapering funnel portion 58 a and the well 173described below. The analysis device 16 also includes an on/off button62, a speaker 64, a handle storage pocket 66, a USB port 68 and a DCinput power port 70 (see FIG. 8). The analysis device 16 also includes abattery 72, as shown in FIG. 9 and a battery door 156 shown in FIG. 11.The battery is preferably rechargeable, however this is not alimitation.

Shroud 56 is the interface between the analysis cartridge 14 and theanalysis device 16. In use, after a breath sample is taken using theanalysis cartridge 14, the user places the analysis cartridge in theanalysis pocket 58 (through pocket opening 58 b) and closes the door 54.FIG. 10 shows the analysis device 16 with the bottom half of the case 48b removed. As shown, the analysis device 16 includes the main circuitboard 74 and the rotation assembly 76 for mixing the elution solution 34and aldehydes, as described below. The rotation assembly 76 alsoincludes the optical system 77. The main circuit board 74 (mother board)is the controller and includes (or is in electrical communication with),but is not limited to, the USB port 68, DC input power port 70, cable(s)for communicating with a motor 78 and the optical system 77 (and opticsboards 160 and 162 described below), a cable for communicating with thehandle assembly 12 (via plugs 107 and 148, cable 106 and circuit board108), the display 60, on/of switch 62, battery 72, and optical sensorsfor sensing if the door 54 is open or closed.

FIG. 11 shows the analysis device 16 exploded and illustrates the firstand second halves of the case 48 a and 48 b, the slideable door 54, themain circuit board 74 and the rotation assembly 76. As shown, theanalysis device 16 also includes an end cap 146 having a plug 148therein for connection of the cable 106, and a tube 150 that defines thehandle storage pocket 66. As discussed above, the handle storage pocket66 and tube 150 include a magnet 150 a that interacts with magnet 111 inthe handle assembly 12. The interaction of the two magnets helps holdthe handle assembly 12 in the handle storage pocket 66. In a preferredembodiment, the first half of the case 48 a includes an opening 152 thataligns with the shroud 56 to define the analysis pocket 58.Opening/cover 154 in the case 48 houses the display 60. In a preferredembodiment, the second half of the case 48 b includes the bottom 57,battery door 156 and openings for the USB port 68 and DC input powerport 70, which are part of the main circuit board 74.

FIGS. 12-20 show the rotation assembly 76 with most other componentsomitted. As shown in FIGS. 12-13, the rotation assembly 76 generallyincludes a rotatable portion 86, first and second fixed members 88 a and88 b, motor 78, a gear train 81 (that preferably includes a pinion gear83 that drives a large gear 84), the shroud 56, and the optical system77. Motor 78 includes a drive shaft (not shown) that drives pinion gear83, which meshes with and rotates large gear 84. Motor 78 is preferablycontrolled by main circuit board 74. It will be appreciated that thecenter portion (the rotatable portion 86) pivots, and the fixed members88 a and 88 b stay stationary within the case 48. Large gear 84preferably includes an arcuate slot 186 therein that receives a guideprotrusion 188 on fixed member 88 a. The ends of the arcuate slot 186provide stops (by interacting with guide protrusion 188) so that therotatable portion 86 can only rotate a certain degree in each direction.

FIGS. 16-17B show the rotatable portion 86 alone. The rotatable portion86 includes first and second axle members 164 and 166 that connect tothe shroud via a key and keyway relationship. This is not a limitation.In another embodiment, the first and second axle members 164 and 166 canbe glued or bonded to the shroud or can be unitary with the shroud. Asshown in FIG. 13, in a preferred embodiment, the shroud 56 includesaxially aligned cylindrical protrusions 174 with a key 56 a on oppositesides thereof. The protrusions 174 receive first and second axle members164 and 166 that include complementary keyways 176 defined therein.First and second axle members 164 and 166 are preferably keyed so thatthey only fit on the shroud 56 in one orientation.

The first and second axle members 164 and 166 have bearings 178 thereonthat cooperate with central openings 180 in the fixed members 88 a and88 b and allow the rotatable portion 86 to rotate. The rotatable portion86 is connected to the large gear 84 via at least one key 166 a thatmeshes with at least one keyway 84 a in the center opening of the largegear 84. The second axle member 166 also includes a stop 184 for thelarge gear 84 and a cable passing recess 165 that allows a cable (notshown) coming from the main circuit board 74 and extending to theoptical system 77 to pass therethrough. Therefore, the motor 78 drivesthe pinion gear 83, which drives the large gear 84, which meshes withand drives the second axle member 166, which drives the shroud 56 (whichholds the analysis cartridge 14 in the analysis pocket 58) and all othercomponents attached thereto, such as the optical system 77 and an arm 80(discussed below).

In a preferred embodiment, the analysis device 16 includes a door lockassembly where the door 54 is locked by the motion of the rotationassembly 76. Preferably, the door lock assembly includes a doordetection sensor that senses whether the door is open or closed. Theshroud 56 includes a cam feature 114 (see FIG. 16) thereon thatinteracts with a pivotal tab member on the case. The cam feature 114 ispositioned so that when the rotation assembly 76 goes to the loadposition the tab is disengage, thus allowing the door to slide open. Inall the other orientations of the rotation assembly 76, the tab is upand that locks the door and prevents it from sliding open.

As will be described below, the rotatable portion 86 rotates with theanalysis cartridge 14 therein to mix the PD solution 35 therein and toallow the optical system 77 to perform its analysis. In addition, therotatable portion 86 includes a cam and lever system to translate therotational motion to pivotal motion, so that the arm 80 pushes thefilter assembly 19 from the breath chamber 30 into the fluid chamber 32.FIGS. 17A-19C show the cam and lever system and how the rotationassembly 76 moves the arm 80. The arm 80 is pivotal on post 191 thatdefines a pivot axis and includes a first end 80 a with a ball bearing122 thereon that rides on a cam surface 120 and a second end that movesin and out of an arm opening 56 b in the side of shroud 56. In apreferred embodiment, the arm 80 is pivotal on post 191 and is securedto a tab 192 that extends from the first axle member 164, as shown inFIG. 17B. Preferably, the arm 80 includes an opening 189 that receivespost 191, which extends downwardly from the shroud 56 (see FIG. 17B).Spring 124 is formed so that the coil section forms an opening thatreceives post 191. A fastener 190 extends through the opening 192 a intab 192 and into the end of post 191. The first end 80 a of the arm 80with the ball bearing 122 extends into a cam channel 194 defined insecond fixed member 88 b (see FIG. 18).

The arm 80 is movable between a stowed position (FIG. 19A) and adeployed position (FIG. 19B). Spring 124 biases the arm to the stowedposition. As shown in FIG. 18, the cam surface 120 is curved andincludes an increasing radius along the path that the ball bearing 122travels. As the rotatable portion 86 (and arm 80) rotates, the ballbearing 122 rides on cam surface 120. The increasing radius of the camsurface 120 causes the arm 80 to pivot about pivot axis and thereforepush the second or working end 80 b of the arm 80 into arm opening 56 bin the shroud 56. Compare FIG. 19A where the arm is not pivoted inwardlyto FIG. 19B where it is pivoted inwardly. Due to the positioning of theanalysis cartridge 14 within shroud 56, the second end 80 b of the arm80 pushes the filter assembly 19 from the breath chamber 30 to the fluidchamber 32. The cam surface 120, ball bearing 122, pivotal (and springbiased via spring 124) arm 80 work together to convert rotational motioninto pivotal motion.

FIGS. 17B-17D and 20 show the optical system 77. The optical system 77includes a housing 196 comprised of first and second halves 197 a and197 b (the second half 197 b is omitted in FIG. 20). The housing 196 ispreferably secured to the shroud 56 by threaded fasteners 196 a (seeFIG. 10). In a preferred embodiment, the shroud 56 includes fourfastener receiver members 175 that receive the elongated threadedfasteners 196 a that extend through complementary first and secondreceiver tubes 177 a and 177 b on the first and second housing halves197 a. The fastener receiver members 175 can be internally threaded orcan be made so that the threads are created in the plastic as thethreaded fastener 196 a is screwed therein. As shown in FIG. 17B, anextra set of first and second receiver tubes 177 a and 177 b areincluded that do not correspond to a fastener receiver member 175 on theshroud 56. The threaded fasteners 196 a therefore secure the two halvesof the housing together and secure the housing 196 to the shroud 56.

Shroud 56 includes an analysis opening 200 in the bottom thereof throughwhich the back of the analysis cartridge 14 extends when it is in theanalysis pocket 58. The analysis opening 200 is aligned with a well 173in the second half 197 b of the housing 196 that receives the back ofthe analysis cartridge 14 therein. The housing 196 includes an analysiscartridge receiving portion 204, as shown in FIG. 17C that defines thewell 173. The housing 196 is formed such that the first and secondhousing halves 197 a and 197 b cooperate to define an light chamber 198,a fluorescence chamber 207, a light trap 94, and the well 173. Theshroud 56 includes three recesses 203 on the bottom surface thereof thatcooperate with recesses on the analysis cartridge receiving portion 204to define a light entry aperture 216, a fluorescence aperture 217 and alight trap opening 218.

The optical system 77 also includes a first optics circuit board ormicrocontroller 162 that includes an LED 79 and a second optics circuitboard 160 that includes a receiver or detector 82 (e.g., a photo diode).Both optics circuit boards include sockets or connectors 163 forconnecting cables (not shown) for communication and control from themain circuit board 74. The optical system 77 also includes at least afirst lens 90 and at least a first filter 92 positioned in the lightchamber 198, and at least a second lens 96 and at least a second filter98 that are positioned in the fluorescence chamber 207. The housing 196is formed such that the first and second housing halves 197 a and 197 bcooperate to define a first lens pocket 199, a first filter pocket 201,a second lens pocket 203 and a second filter pocket 205.

As shown in FIGS. 17C-17E and 20, the top housing half 197 a includesfirst and second recesses 212 and 214 defined therein that cooperatewith first and second recesses 213 and 215 in the top surface of theanalysis cartridge receiving portion 204 to at least partially form thelight entry aperture 216 and the fluorescence aperture 217. The top andbottom housing halves 197 a and 197 b also cooperate to at leastpartially form the light trap opening 218. When the analysis cartridge14 is positioned in the well 173, the light entry window 172 a isaligned with the light entry aperture 216, the fluorescence window 170is aligned with the fluorescence aperture 217, and the light exit window172 b is aligned with the light trap opening 218.

In use, the LED 79 shines light along a light path (LP) through thefirst lens 90, through a first filter 92, through light entry aperture216, through light entry window 172 a in analysis cartridge 14 (where itcauses the CCM in the fluorescing solution 206 to fluoresce within thesensing chamber 32 b), through light exit window 172 b, through lighttrap opening 218 and into light trap 94. The light trap 94 is configuredwith angled walls so that the light that enters therein bounces aroundand cannot escape back through the entry opening and be reflected in anyway toward the detector 82. The light reflected from the fluoresced CCMexits the analysis cartridge 14 along a fluorescence path (FP) throughfluorescence window 170 at an approximately 90 degree angle from thelight entering the analysis cartridge 14. The fluorescence path travelsthrough fluorescence aperture 217, through the second lens 96 and thesecond filter 98 and to the detector 82. This is generally an emitterdetector set-up. In a preferred embodiment, the detector 82 is at aboutninety degrees to the emitter 79. Other angles are within the scope ofthe invention.

In a preferred embodiment, the light emitted from the LED 79 anddirected along the light path LP is as collimated as possible.Preferably, the light chamber 198 is designed to eliminate as much lightas possible that is not collimated. To accomplish this, the lightchamber 198 includes at least the first lens 90, and a series of bafflesand apertures (described below) positioned in the light path LP. FIG.17D shows the light path LP as being directed parallel to the axis ofthe light chamber 198. However, some light emitted from the LED 79 maynot extend parallel to the axis. See, for example, the dashed lines inFIG. 17D. In a preferred embodiment the first lens 90 is a Fresnel lens.However, this is not a limitation on the present invention. In anexemplary embodiment, the first lens 90 is a Fresnel lens with a focallength of about 10 mm, overall dimensions of 25.4 mm×25.4 mm×22 mm witha lens diameter of 12.7 mm (the second lens can have the sameproperties). However, none of these dimensions are limiting. The firstlens 90 is positioned and includes specifications such that itpreferably focuses the light from the LED 79 inside of the sensingchamber 32 b, within the well 173. In an exemplary embodiment, thecollimated beam of light is approximately 4 mm in diameter in the centerof the sensing chamber 32 b.

As shown in FIG. 17C-17D, in a preferred embodiment, the light chamber198 includes a first light baffle 244 positioned therein that includes afirst light baffle aperture 244 a defined therein. The first lightbaffle 244 is positioned along a light path LP before the first lens 90.The light chamber 198 also includes a second light baffle 246 positionedtherein that includes a second light baffle aperture 246 a definedtherein. The second light baffle 244 is positioned along the light pathLP between the first lens 90 and the first filter 92. Preferably, athird light baffle 248 that includes a third light baffle aperture 248 ais positioned in the light path LP after the first filter 92.Preferably, the first and second light baffles are orthogonal to thedirection of the light path LP and the third light baffle is notorthogonal to the direction of the light path LP. It will be appreciatedthat the baffles and apertures are formed by the first housing half 197a cooperating with the second housing half 197 b. FIG. 17D only showsthe second housing half 197 b.

After the light passes through the analysis cartridge passes through thelight trap opening 218 and into the light trap 94. In a preferredembodiment, the walls of the light trap are black, which will absorb themajority of light that enters. FIG. 17D shows a plan view of the lighttrap 94. In a preferred embodiment, the light trap 94 includes curvedwalls that help disperse the light as it bounces off. However, as shownin FIG. 17D, with respect to the light path LP, the walls form anglesthat are designed to absorb most light because they are black, but alsoto reflect any light that is reflected toward another wall so thatvirtually no light escapes back through the light trap opening 218. Thelight trap 94 preferably includes a first wall 94 a that receives thelight after it enters the light trap. The first wall is preferablyangled between about 25° and about 45° with respect to the light pathLP. In an exemplary embodiment it is angled at 35° from the light pathLP. The light trap 94 also preferably includes a second wall 94 b thatis angled from the light path LP. Preferably, it is not at a right anglewith the light path LP. In an exemplary embodiment, as light enters thelight trap 94, every time it bounces off a different wall approximately80% is absorbed and 20% is reflected. After bouncing off of a few wallswith the 80% to 20% absorption to reflection ratio, the remaining lightwill be negligible.

The fluorescence path FP also includes baffles and apertures thereintogether with the second lens 96 and second filter 98. As shown in FIG.17D, in a preferred embodiment, the fluorescence path FP includestherealong a first fluorescence baffle 250 and related aperture, whichis the fluorescence aperture 217 that is formed by the second recess 215in the top surface of the analysis cartridge receiving portion 204 andthe defined therein, the second lens 96, a second fluorescence baffle252, which includes a second fluorescence baffle aperture 252 a definedtherein and second filter 98. Preferably, the second lens 96 has thesame specifications as the first lens 90. However, this is not alimitation and the two lenses can be different. In another embodimenteither or both of the light path and the fluorescence path can includemore than one lens therein.

It will be appreciated that the light emitted from the LED is notcompletely collimated. Therefore, the light baffles and apertures areprovided to block some of the light that is reflected off of the insideof the light chamber 198 and other extraneous light. The apertures inthe first, second and third light baffles 244, 246 and 248 have smallerdiameters than the light chamber 198, thereby causing the light bafflesto block or eliminate non-collimated light some light and help create amore collimated beam traveling along the light path LP through the lightchamber 198.

In a preferred embodiment, the diameters of the first, second and thirdlight baffles get smaller as they are encountered along the light path.In an exemplary embodiment, the light chamber 198 has an inner diameterof about 16 mm, the first light baffle aperture has an inner diameter ofabout 10 mm, the second light baffle aperture has an inner diameter ofabout 9 mm, and the first light baffle aperture has an inner diameter ofabout 5 mm. As for the fluorescence chamber 207, in an exemplaryembodiment, the first fluorescence baffle aperture has an inner diameterof about 5 mm and the second fluorescence baffle aperture has an innerdiameter of about 7 mm.

As shown in FIG. 17D, in a preferred embodiment, the first lens pocket199, first filter pocket 201, second lens pocket 203 and second filterpocket 205 each include crush ridges therein that help maintain the lensor filter therein in a stable position and prevent it from vibrating.However, the crush ridges can be omitted. Also, the well 173 can includean alignment member 254 therein and a drain 256 for draining any fluidin the well 173.

The first filter 92 is provided to filter unwanted wavelengths of lightfrom the beam of light emitted from the LED 79. Any filter is within thescope of the present invention. In a preferred embodiment, the firstfilter allows transmission of light in a first range. For example, thefirst filter can include a transmission region of 300 nm to 540 nmT<0.0001%, OD>6, a transition region 540 nm to 550 nm, 0%<T<100%, and ablocking region that blocks light between 550 nm and 800 nm, T>90%.However, this is only exemplary and any filter is within the scope ofthe present invention. In a preferred embodiment, the second filterallows transmission of light within a second range. For example, thesecond filter can include a blocking region that blocks light between300 nm and 555 nm, T<0.0001%, OD>6, a transition region 555 nm to 565nm, 0%<T<100%, and a transmission region of 565 nm to 800 nm T>90%.However, this is only exemplary and any filter is within the scope ofthe present invention. The second filter 98 is designed to block all LEDlight that somehow made it into the fluorescence chamber 207 and to onlyallow the fluorescent light through at a predetermined wavelength (e.g.,a long pass filter).

In a preferred embodiment, to further separate the fluorescence signals,lock-in amplification is used. A lock-in amplifier is used to helpeliminate signals that have an origin in background light (e.g., lightsfrom the room, circuit boards inside the device, backlighting from thescreen, and any other white source that could potentially reach thedetector 82). In an exemplary embodiment of using this technique, theLED is blinked on and off at a first rate (e.g., between 400 HZ and 1000HZ). This helps get away from DC, which helps the noise issues. Then, ifwhile detecting a signal is detected that does not have the samefrequency and is very close in phase to the frequency at which the LEDis being driven, there is a likelihood that the light is coming fromsome other source (e.g., background light), so it is eliminated from thesignal. Generally, the lock-in amplifier takes the signal, it averagesthe signal from when the LED is on and then it averages the signal fromwhen the LED is off and it subtracts the two. This preferably results ina fluorescence signal with little noise.

As shown in FIG. 17C, In a preferred embodiment, the upper housing half197 a includes a lip or flange 258 that extends downwardly and overlapsanother lip or flange 260 extending upwardly from the lower housing half197 b. The complementary flanges help block light for entering orexiting the light chamber 198 or the fluorescence chamber 207.Preferably, the flanges are offset from one another so that theyoverlap.

As discussed herein, the components of the optical system 77 are tunedspecifically for the chemistry of an analysis cartridge and for thisparticular PD derivative and the amount of fluorescence that is to bemeasured. The ninety degree angle allows the photo detector 82 to detectthe emitted light from the fluoresced CCM. In other words, the detector82 is not receiving any light from the LED, but is only detecting theparticles of aldehyde that get fluoresced.

From the description herein, it should be understood that the breathanalysis system is used preferably to obtain a breath sample from apatient in the analysis cartridge 14 and then the breath sample isanalyzed in the analysis device 16. Once the analysis cartridge 14 isplaced in the analysis pocket 58 in the analysis device 16 and extendsdown into the well 173, the rotation assembly 76 rotates the analysiscartridge 14 a number of times to mix the contents, to move the filterassembly 19 from the breath chamber 30 to the fluid chamber 32 and tolet the optical system 77 perform an analysis.

FIGS. 21A-23 show the steps for how the rotation assembly 76 mixes thePD solution 35, moves the filter assembly 19 from the breath chamber 30to the fluid chamber 32, mixes the PD solution and breath aldehydes(CCM) to form a fluorescing solution 206 and how the optical system 77performs an analysis of the fluorescing solution 206. Each of thefigures shows a cross-sectional end elevation view of the rotationassembly 76 with the analysis cartridge 14 in the analysis pocket 58. Ina preferred embodiment, as shown in FIG. 21A, when the analysiscartridge is placed in the analysis pocket 58 and the back extendsthrough the analysis opening 200 down into the well 173, an alignmentmember 208 is received in the breath exit hole 40.

In FIG. 21A, the analysis cartridge 14 is in the analysis pocket 58 inthe rotation assembly 76 and is in the configuration as the clinicianhas just taken it off the handle assembly 12 and placed it in theanalysis pocket 58 (referred to herein as the start position). In use,the rotation assembly 76 rotates the analysis cartridge 14 through atleast one pivot or rotation to introduce or mix the PD derivative 24into the elution solution 34. FIG. 21B shows the orientation of therotatable portion 86 and the analysis cartridge 14 in a second positionafter rotation (referred to herein as a first mixing position). Therotatable portion 86 can move between the start position and the firstmixing a predetermined number of times for proper stirring or mixing. Asdiscussed above, the rotatable portion 86 is moved via motor 78 (seeFIG. 15), which rotates the rotatable portion 86 and basically sloshesthe PD solution 35 back and forth to make sure that the PD derivative 24is completely in solution. This is the mixing step.

FIG. 21C shows the orientation of the rotatable portion 86 and theanalysis cartridge 14 in a third position (referred to herein as abaseline reading position). At this step (referred to herein as abaseline reading step), the analysis cartridge 14 is pivoted so that thefluid chamber 32 is straight up and down. Therefore, all of the PDsolution 35 is in the rear portion or sensing chamber 32 b of the fluidchamber 32. At this point a baseline fluorescence reading is taken bythe optical system 77. In a preferred embodiment, this is done byturning on the LED 79 (the main circuit board 74 communicates with thefirst optical circuit board 162) and measuring the fluorescence of thePD solution 35 without any CCM therein, using detector 82. The baselinereading is communicated by the second optical circuit board 160 to themain circuit board 74.

Next, as shown in FIGS. 21D-21E, the rotatable portion 86 and shroud 56(and the analysis cartridge) move through and past the first mixingposition (shown in FIG. 21D) and to a fourth position shown in FIG. 21E(referred to herein as the insertion position), where the arm 80 pusheson the filter assembly 19 and moves it along the filter assembly path P2(see FIG. 2) from the breath chamber 30 to the fluid chamber 32. Inother words, in this step, the filter assembly 19 is inserted into thefluid chamber 32 by the arm 80. When this happens, the second breakablebarrier 36 b is broken. It will be appreciated that the arm 80 pushesfilter assembly 19 as a result of the cam path 120 discussed above. Asshown in FIG. 21D, the arm 80 is still in the stowed position after thebaseline reading step. Therefore, during the mixing step and thebaseline reading step and the rotation between the start position, thefirst mixing position, and the analysis position, the cam path 120 isconfigured such that the arm 80 remains in the stowed position. However,when the rotatable portion 86 rotates beyond the first mixing position,the increasing radius of the cam surface 120 pushes the ball bearing 122outwardly, thereby pivoting the second end 80 b of the arm 80 andpushing the filter assembly 19 inwardly, as shown in FIG. 21E. In thisposition, all of the fluid is down in the front portion 32 a of thefluid chamber 32 and the filter assembly 19 (frit plates 26 andsubstrate 28) is now in the fluid chamber 32. However, the PD solution35 has not yet touched any of the frit plates 26 or substrate 28 becauseof the fluid volume.

Next, as shown in FIG. 22, the rotatable portion 86 and shroud 56 (andthe analysis cartridge) rotate to a fifth position (referred to hereinas the analysis position), where the fluid chamber 32 is once againstraight up and down. It will be appreciated that the positioning of therotatable portion 86 is the same in the analysis position and thebaseline reading position. In this position, the PD solution 35 filtersthrough opening 17 in the frit stack holder 20 and the frit plates 26and the substrate 28 thereby immersing the frit plates 26 and the silica28 in the PD solution 35. Also, the arm 80 has retracted back to thestowed position, but the filter assembly 19 has stayed in place. As thePD solution drains down and drips through the frit plates 26 it washesthe CCM off the substrate 28 and into solution (referred to herein asthe fluorescing solution 206). The analysis cartridge 14 is left in thisorientation for a predetermined amount of time; enough time for the PDsolution 35 to drain through and collect in the sensing chamber 32 b ofthe fluid chamber 32 (the drainage step). During this step, the CCM arelabeled or painted with the PD solution. In another embodiment, anothermixing step can be added to further mix the fluorescing solution. Next,a fluorescence reading is taken by the optical system 77 to analyze thefluorescing solution. The original reading was the baseline without anyCCM in the solution and now a measurement with CCM is taken.

After the analysis step, the rotation assembly 76 rotates to a sixthposition, which is the same as the first position. In other words, therotatable portion 86 returns to the start position so that the analysiscartridge 14 can be removed, as shown in FIG. 23. The analysis cartridge14 can then be disposed. In a preferred embodiment, all the stepsdescribed above are done automatically. Basically, the user opens thedoor 54, puts the analysis cartridge 14 in, closes the door 54 and hitsgo or the like on the display 60.

FIGS. 24-29 show another embodiment of the present invention where someof the steps discussed above with the analysis cartridge 14 are dividedinto system that includes a breath analysis cartridge 220 and afluorescence analysis cartridge 222. The two cartridges together arereferred to herein as an analysis cartridge system 219. The structure ofboth the breath analysis cartridge 220 and a fluorescence analysiscartridge 222 is similar to the analysis cartridge 14 described above sothat they can fit into the analysis pocket 58, as described below. Likenumerals in FIGS. 24-29 refer to like components in FIGS. 1-23.

The analysis cartridge system 219 is used to capture breath aldehydes(CCM) and analyze them with the optical system 77 in the device similarto the breath cartridge and system described above. The general steps inusing the analysis cartridge system 219 are as follows: 1) blow throughthe breath chamber 30 in the breath analysis cartridge 220 to captureCCM; 2) place the breath analysis cartridge 220 in the analysis pocket58; 3) allow the rotation assembly 76 and arm 80 to push move the filterassembly 19 from the breath chamber 30 to the fluid chamber 32 where theCCM mix with the elution solution 34 to form a CCM solution; 4) removethe breath analysis cartridge 220 from the analysis pocket 58; 5) movethe ampule member from the first position to the second position toallow the CCM solution to mix with the PD derivative to form painted CCMsolution 209; 6) connect the breath analysis cartridge 220 to thefluorescence analysis cartridge 222 so the painted CCM solution drainsinto the upper chamber of the fluorescence analysis cartridge 222 andthrough the filter assembly 19. The substrate 28 in the filter assembly19 captures the painted CCM from the painted CCM solution 209 and theabsorption member 238 absorbs the remaining solution; 7) place thefluorescence analysis cartridge 222 in the analysis pocket 58; 8) allowthe rotation assembly 76 and arm 80 to move the filter assembly 19 fromthe breath chamber 30 to the fluid chamber 32 where the painted CCM iseluted into a second elution solution 202 to form the fluorescingsolution 206; 9) perform a fluorescence detection analysis of thefluorescing solution 206 with the optical system 77. In a preferredembodiment, the second elution solution rinse comprises greater than 50%acetonitrile and preferably 90% ethanol.

As shown in FIG. 25, the breath analysis cartridge 220 includes an upperor breath chamber 30, a fluid chamber 32 and a filter assembly 19 (withsubstrate 28 therein). A cap 221 plugs and seals the fluid chamber 32.Breath analysis cartridge 220 includes an ampule assembly 224 positionedin the back of the fluid chamber 32. Elution solution 34 is disposed inthe fluid chamber 32, and the fluid chamber 32 is sealed from the breathchamber 30. This can be done by a breakable foil barrier, as describedabove or by another sealing method. For example, the frit stack holder20 can seal the filter assembly opening 134. In use, a patient blowsthrough breath chamber 30 so that breath aldehydes are collected on thesubstrate 28. Next, the breath analysis cartridge 220 is placed in theanalysis pocket 58 in the start position (see FIG. 21A). Then therotation assembly 76 rotates the shroud 56 and breath analysis cartridge220 to the insertion position (see FIG. 21E) so that the filter assembly19 moves from the breath chamber 30 to the fluid chamber. In the fluidchamber the CCM are eluted into the elution solution 24 to form the CCMsolution. The breath analysis cartridge 220 is then removed from theanalysis pocket 58.

In a preferred embodiment, the ampule assembly 224 includes an ampulemember 226 that is received in and slidable within a slide tube 228. Theampule member 226 is preferably a cylinder that defines an interior 226a, includes enclosed ends and has at least one and preferably two fluidopenings 230 defined in the sidewall thereof. The end that protrudesoutside of the fluid chamber is enclosed with a breakable barrier 231.As shown in FIG. 25, the PD derivative 24 is disposed in the ampulemember interior or fluorescence chromophore space 226 a. The ampulemember 226 is movable within the slide tube 228 between a first positionwhere the PD derivative 24 are separated from the fluid chamber 32 and asecond position where the ampule member interior 226 a is incommunication with the fluid chamber 32. In a preferred embodiment, whenthe ampule member 226 is in the first position, the fluid openings 230are positioned inside the slide tube 228 and are therefore sealed fromallowing the elution solution 34 therein, as shown in FIG. 25. However,when the ampule member 226 is slid to the second position, the openings230 are now in flow communication with the fluid chamber 32, whichallows fluid in the fluid chamber 32 to enter the ampule member interior226 a. FIG. 26 shows the ampule member 226 in the second position. Asshown in FIGS. 24-25, in a preferred embodiment, the ampule assembly 224is housed in a receiver member 232 that mates with the fluorescenceanalysis cartridge 222 as described below. In use, after removing thebreath analysis cartridge 220 from the analysis pocket 58, as describedabove, the user presses the ampule member 226 and moves it from thefirst position to the second position.

As shown in FIG. 27, in a preferred embodiment, the fluorescenceanalysis cartridge 222 includes an upper chamber 30, a fluid chamber 32and a filter assembly 19 (with substrate 28 therein). The second elutionsolution 202 is disposed in the fluid chamber 32. A cap 221 plugs andseals the fluid chamber 32. A piercing member 234 is disposed in theupper chamber 30 adjacent the front opening 33. The piercing member 234is a hollow tubular member that includes a main body portion 235 with apiercer 236 extending therefrom. The piercer 236 has a smaller diameterthan the main body portion and the upper chamber 30.

In use, as shown in FIGS. 28-29, the receiver member 232 of the breathanalysis cartridge 220 is inserted into the front opening 33 of thefluorescence analysis cartridge 222. The piercer 236 then pierces thebreakable barrier 231 thereby communicating the ampule member interior226 a with the lower chamber 32 of the fluorescence analysis cartridge222. When the breakable barrier 231 is pierced, the painted CCM solution209 flows into the upper chamber 30 of the fluorescence analysiscartridge 222 and washes over the filters 26 and substrate 28 in thefilter assembly 19 and the painted CCM are captured by the substrate 28.Any excess solution is absorbed in the absorption member 238 in the rearof the upper chamber 30.

The fluorescence analysis cartridge 222 is then placed in the analysispocket 58 in the analysis device 16 in the start position (see FIG.21A). The rotation assembly 76 then rotates the fluorescence analysiscartridge 222 to the insertion position (see FIG. 21E) where the paintedCCM is eluted into the second elution solution 202 to form thefluorescing solution 206. The rotation assembly 76 then rotates thefluorescence analysis cartridge 222 to the analysis position (see FIG.22) and a fluorescence analysis of the fluorescing solution 206, asdescribed above, is performed.

FIG. 30 shows another embodiment of an analysis cartridge 240. Thestructure of analysis cartridge 240 is similar to the analysis cartridge14 described above so that they can fit into the analysis pocket 58, asdescribed below. Like numerals in FIG. 30 refer to like components inFIGS. 1-29. As shown in FIG. 30, the analysis cartridge 240 includes anupper chamber or breath chamber 30, a fluid chamber 32, a filterassembly 19 (with filters 26 separated by a substrate space 27) and avent cap 242 to seal the fluid chamber 32. Substrate that is pre-loadedwith the PD derivative (referred to herein as PD substrate) is disposedin substrate space 27 and elution solution 34 is disposed in the fluidchamber 32.

In use, the analysis cartridge 240 is placed on the handle assembly 12,a user blows through the mouthpiece 18 and the breath chamber 30 for apredetermined amount of time and at a predetermined pressure (or withina predetermined pressure range) until CCM are collected on the PDsubstrate 241. After the CCM have been collected, the user removes theanalysis cartridge 240 from the handle assembly 12, removes themouthpiece 18 and places the analysis cartridge 240 in the analysisdevice 16.

At first, the analysis cartridge 240 is in the analysis pocket 58 in thestart position (see FIG. 21A). In a preferred embodiment, no firstmixing or baseline reading step is needed. However, in anotherembodiment, these steps can be included. The rotation assembly 76 thenrotates the analysis cartridge 14 through and past the position shown inFIG. 21D and to the insertion position where the arm 80 pushes on thefilter assembly 19 and moves it along the filter assembly path P2 fromthe breath chamber 30 to the fluid chamber 32. In other words, in thisstep, the filter assembly 19 is inserted into the fluid chamber 32 bythe arm 80. It will be appreciated that the arm 80 pushes filterassembly 19 as a result of the cam path 120 discussed above. When therotatable portion 86 rotates to the insertion position the increasingradius of the cam surface 120 pushes the ball bearing 122 outwardly,thereby pivoting the second end 80 b of the arm 80 and pushing thefilter assembly 19 inwardly, as shown in FIG. 21E. In this position, allof the fluid is down in the front portion 32 a of the fluid chamber 32and the filter assembly 19 is now in the fluid chamber 32. However, theelution solution 34 has not yet touched any of the frit plates 26 or thePD substrate 241 because of the fluid volume.

Next, as shown in FIG. 22, the rotatable portion 86 and shroud 56 (andthe analysis cartridge) rotate to the analysis position, where the fluidchamber 32 is once again straight up and down. In this position, theelution solution 34 filters through opening 17 in the frit stack holder20 and the frit plates 26 and the PD substrate 241 thereby immersing thefrit plates 26 and the PD substrate 241 in the elution solution 34. Asthe elution solution 34 drains down and drips through the frit plates 26it washes the painted CCM off the PD substrate 241 and into solution(referred to herein as the fluorescing solution 206). The analysiscartridge 240 is left in this orientation for a predetermined amount oftime; enough time for the elution solution 34 to drain through andcollect in the sensing chamber 32 b of the fluid chamber 32 (thedrainage step). In another embodiment, another mixing step can be addedto further mix the fluorescing solution. Next, a fluorescence reading istaken by the optical system 77 to analyze the fluorescing solution.

After the analysis step, the rotation assembly 76 rotates back to thestart position so that the analysis cartridge 240 can be removed, asshown in FIG. 23. The analysis cartridge 240 and can then be disposed.

The analysis device 16 and the screen 60 thereof includes the ability towalk a patient and the practitioner through the steps necessary toperform a breath analysis. For example, the screen provides a user withfeedback on, for example, the flow rate to let a patient know if theyare blowing too hard or too soft.

An exemplary set of steps using the system 10 and the user interface(UI) on the screen 60 is described below. It will be understood thatthis is only exemplary and that steps can be rearranged and/or omittedor added as desired. Furthermore, it will be understood that all entriesare being made on the UI. In another embodiment, the UI buttons andkeypad can be manual buttons and a keypad. The practitioner steps are asfollows: 1) Turn on the device by pressing the power button 62 locatedabove the touchscreen. 2) Press the start button on the UI. 3) Press thelist button on the bottom left of the UI keypad and select thepractitioner name in the upper right corner. This auto-populates thePractitioner ID. Press the go button. 4) Enter the Patient ID in thePatient ID field (could be a number or the patient email address). Pressthe go button. 5) Enter the 6-digit lot number in the analysis cartridgelot number field. Press the go button. 6) Open the analysis cartridgepackage. 7) Remove the handle assembly 12 from the handle storage pocket66. Press the arrow button “>” to continue. 8) Give the breath captureassembly 13 to the patient. Press the arrow button “>” to continue. 9)Press start.

The patient steps are as follows. 1) Press “tap to start”. 2) Deliverbreath sample by blowing through the mouthpiece 18, keeping the circlein the green zone on the UI. A ring will appear on the outside of thecircle that represents the total volume. Maintain the green circle untilthe ring shows 100% complete. 2) When 100% total volume is reached, stopblowing and give handle to practitioner.

Continued practitioner steps are as follows. 10) Press “next”. 11)Disconnect the analysis cartridge 14, 220 or 240 from the handleassembly 12. Press the arrow button “>” to continue. 12) Place thehandle assembly back in the handle storage pocket 66. Press the arrowbutton “>” to continue. 13) Disconnect the mouthpiece 18 from theanalysis cartridge 14, 220 or 240. Press the arrow button “>” tocontinue. 14) Open the door 54. Press the arrow button “>” to continue.Insert the analysis cartridge 14, 220 or 240 into the analysis pocket58. Press the arrow button “>” to continue. 15) Close the door 54. 16)Press “done”. 17) The analysis device 16 will begin processing thesample. When it is finished, it will display “100%.” 18) Tap to revealthe score. 19) Press “done”.

It will be appreciated that if the analysis cartridge system 219 isused, the final few steps will change. After step 16 the steps are asfollows. 17) The analysis device 16 will push the filter assembly 19 inthe breath analysis cartridge 220 to the second position. When it isfinished, it will display “done.” 18) Open the door 54 and remove thebreath analysis cartridge. 19) Press ampule member. 20) Connect breathanalysis cartridge to fluorescence analysis cartridge to allow thepainted CCM solution to enter fluorescence analysis cartridge. 21)Disconnect breath analysis cartridge from fluorescence analysiscartridge. 22) Insert the fluorescence analysis cartridge 222 into theanalysis pocket 58. Press the arrow button “>” to continue. 23) Closethe door 54. 24) Press “done”. 25) The analysis device 16 will beginprocessing the sample. When it is finished, it will display “100%.” 26)Tap to reveal the score. 27) Press “done”.

If the device is connected to Wi-Fi, the device will automaticallyupload the test record to a portal. If not, it will store the scoreuntil it finds a secure connection. The score can also be uploaded viaUSB port 168.

It will be appreciated that modifications to the invention can be made.For example, the mouthpiece can be non-removable,

The present invention is directed to a method and device useful for thedetection, quantitation and assay of carbonyl containing moieties(“CCM”) including aldehydes, preferably in biological samples, andpreferably at low concentrations in the biological sample. In thisregard, CCM is defined to include one or more different carbonylcontaining moieties.

As used herein, a “biological sample” is referred to in its broadestsense, and includes solid and liquid or any biological sample obtainedfrom nature, including an individual, body fluid, cell line, tissueculture, or any other source. As indicated, biological samples includebody fluids or gases, such as breath, blood, semen, lymph, sera, plasma,urine, synovial fluid, spinal fluid, sputum, pus, sweat, as well asliquid samples from the environment such as plant extracts, pond waterand so on. Solid samples may include animal or plant body parts,including but not limited to hair, fingernail, leaves and so on. Thepreferred biological sample for one embodiment of the present inventionis the breath of a human.

A CCM is a compound having at least one carbonyl group. A carbonyl groupis the divalent group >C=0, which occurs in a wide range of chemicalcompounds. The group consists of a carbon atom double bonded to anoxygen atom. The carbonyl functionality is seen most frequently in threemajor classes of organic compounds: aldehydes, ketones, and carboxylicacids. As used herein, “aldehyde” has its ordinary chemical meaning andthe method of the present invention is useful in detecting theconcentration of aldehydes in biological samples. In particular, thepresent invention is useful in detecting various forms of aldehydesinclude without limitation 1-hexanal, malondialdehyde, 4-hydroxynonenal,acetaldehyde, 1-propanal, 2-methylpropanal, 2,2-dimethylpropanal,1-butanal, and 1-pentanal.

The amount of the CCM captured by the substrate may vary, but typicallyfor a substrate consisting of 200 mg of 50-270 mesh (300-50 μm) particlewith a bed diameter of 12.5 mm, generally, it will be equivalent to theamount in a human's breath after breathing into a tube like abreathalyzer. Preferably it will be from 75 to 0.1 ppb (400 to 4 pmoles)and more preferably from 20 ppb to 0.01 ppb (80 to 0.4 pmoles).

The invention is amenable to “mix & read” and “real-time” assay formatsfor the detection of CCM. The invention can be applied to the detectionof CCM in solution. The invention can be applied to the detection oftrace CCM in the gas phase by the addition of a primary capture (on asubstrate as discussed below) and release (elution from the loadedsubstrate as discussed below) process. Preferably in one step of theprocess, gas phase CCM, for example, aldehydes from the breath of ahuman, are captured on a substrate.

The substrate of the present invention is desirably formed from a solid,but not necessarily rigid, material. The solid substrate may be formedfrom any of a variety material, such as a film, paper, nonwoven web,knitted fabric, woven fabric, foam, glass, etc. For example, thematerials used to form the solid substrate may include, but are notlimited to, natural, synthetic, or naturally occurring materials thatare synthetically modified, such as polysaccharides (e.g., cellulosematerials such as paper and cellulose derivatives, such as celluloseacetate and nitrocellulose); polyether sulfone; polyethylene; nylon;polyvinylidene fluoride (PVDF); polyester; polypropylene; silica;inorganic materials, such as deactivated alumina, diatomaceous earth,MgSO₄, or other inorganic finely divided material uniformly dispersed ina porous matrix, with polymers such as vinyl chloride, vinylchloridepropylene copolymer, and vinyl chloride-vinyl acetate copolymer;cloth, both naturally occurring (e.g., cotton) and synthetic (e.g.,nylon or rayon); porous gels, such as silica gel, agarose, dextran, andgelatin; polymeric films, such as polyacrylamide; and so forth.Preferably the substrate is a solid phase matrix of silica optionallyspaced between frits. The size of the substrate is chosen so that ameasurable amount of CCM is captured by the substrate. The size can varybut generally it is about 2 mL, preferably about lmL and more preferablyabout 0.25 mL.

The substrate typically consists of a bed of particles with 50-60angstrom pores, with a 50-270 mesh (300-50 μm), and a mass of 75 to 300mg, preferably 60-120 mesh (250-125 μm) with a mass of 100 to 200 mg andmore preferably 50-120 mesh (210-125 μm) with a mass of 125 to 175 mg.

In another step of the process, a fluorescence chromophore such as aphenylene diamine derivative is added to an elution solution to form aphenylene diamine solution. Phenylene diamine derivatives useful inconnection with the present invention include but are not limited tomany phenylene diamine derivatives including without limitationmeta-phenylene diamine (“mPDA”) and its derivatives, with mPDA preferredfor detecting aldehydes including without limitation 1-hexanal. Whilecertain p-PDA or o-PDA derivatives may be useful in the method of thepresent invention, they are not useful for detecting 1-hexanal as theyyield a cloudy colloidal suspension which is not useful for the opticalbased detection discussed below.

Other phenylene diamine derivatives include the following or mixturesthereof:

where R1, R2, R3, R4 are each independently selected from the groupconsisting of H, alkyl, substituted alky, alkoxy, substituted alkyoy,acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl,aminothiocarbonyl, aminocarbonylamine, aminothiocarbonylamino,aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino,amidino, carboxyl, carboxyl ester, (carboxylester) amino, (carboxyester) oxy, cyano, halo, hydroxy, SO3-, sulfonyl, substituted sulfonyl,sulfonyloxy, thioacyl, thioal, alkylthio, substituted alkylthio, acyl,substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,substituted cycloalkyl, heterocycles, and substituted heterocycles.

The mPDA derivative mPDA-orange(pyridinium,4-[2-[4-(diethylaminio)phenyl-ethenyl]-1-[1-(3,5-diminobenzamide)-pentylamino-5-oxyhextyl])leverages both a) the sensitivity to environmental changes and b) thepotential to modulate the surfactant dependence of the mPDA-aldehydeinduced polymerization. The scheme used to synthetize mPDA-orange is toconjugate mPDA to the styrylpyridinium moiety via an alkyl amide linker.

mPDA-orange exhibits a quantum yield increase as the molecule isincorporated into the aldehyde induced mPDA polymer. In addition, theexcitation and emission properties of the styrylpyridinium moietyaffords a FRET (Forster Energy Transfer) generated signal from the mPDApolymer. The styrylpyridinium moiety exhibits a broad excitation with amaximum at 470 nm and an emission maximum at 570 nm. The excitationprofile provides sufficient overlap with the emission profile of themPDA polymer to afford FRET based signal generation. A Fret based signalgeneration would be manifest by an excitation at the mPDA polymer (405nm) and emission at the styrylpyridinium moiety emission at 570 nm.

A direct aldehyde induced polymerization of mPDA-orange alone does notgenerate a response signal due to quenching of the styrylpyridinium atthe high concentrations required for induction of the polymer. Aresponse would only be expected when the mPDA-orange is contained withina mixture of mPDA and mPDA-orange. Indeed, an aldehyde response is onlyobserved when mPDA-orange is doped into mPDA at significantly dilutemolar ratios mPDA/mPDA-orange 1,000:1 to 10,000:1. An increase inmPDA-Orange emission at 570 nm is observed when excited at 405 nm when 1μM hexanal is added to the system. The increase in emission is notobserved when the mPDA-orange styrylpyridinium moiety is exciteddirectly at 470-490 nm. The response is approximately 3× over thebackground, where the conditions are 7 mM mPDA, 5 μM mPDA-orange (molarration 15,000:1), 90 mM NaCl, 15% Ethanol, 0.1% SDS, 50 mM citrate at ph2.5. The excitation is at 405 nm and the emission at 575-585 nm. As canbe seen, in the absence of aldehyde the background level remains fairlyconstant and auto induction leading to incorporation of mPDA-orangeappears to be minimal Though the response for mPDA-orange is much less3× versus 15× for mPDA alone the derivative offers severaladvantages: 1) increase wavelength discrimination afforded by thelargeStokes shift between excitation and emission and 2) the enhancedbaseline stability

In general, the concentration of the phenylene diamine derivative in thephenylene diamine solution ranges from 0.5 mM to 25 mM. For mPDA, themPDA concentration in the phenylene diamine solution generally rangesfrom 0.5 to 21 mM, preferably from 2 to 10 mM, and optimally 5 mM foraldehydes such as 1-hexanal. Notwithstanding the foregoing, formPDA-orange, it must be diluted into mPDA at a low molar ration,preferably 1000-10,000.

In general, the elution solution includes a salt, a buffer, asurfactant, and an organic solvent. The concentration of the salt rangescan from 5 mM to 200 mM and preferably from 20 mM to 80 mM; theconcentration of the buffer can range from 25 mM to 200 mM andpreferably from 40 mM to 60 mM; and the concentration of the surfactantcan range from 0.05% (1.7 mM) to 0.4% (13.9 mM), and preferably from0.15% (5.2 mM) to 0.25% (8.7 mM). Optimally 0.2% or 6.96 mM is used. Thesalt can be any salt that does not negatively impact the fluorescingsolution and controls salting effects in the elution solution, and mayinclude NaCl, LiCl, KCl, sulfates and phosphates, and mixtures thereof,with NaCl preferred.

The buffer is employed to maintain the elution solution mildly acidicand preferably at a pH of between 2 and 4.5, more preferably 2.5. Thebuffer can be a borate buffer, a phosphate buffer, a citrate buffer, anorganic buffer such as HEPES (1-piperazineethane sulphonic acid) or alsoa TRIS (tris(hydroxymethyl)aminoethane) buffer, preferably a citratebuffer for use in detecting aldehydes.

The surfactant can include sodium decyl sulfate, sodium dodecyl sulfate(“SDS”), sodium tetradecyl sulfate and Standapol ES-1, with SDSincluding the C10, C12 and C14 version of SDS is preferable. TritionX-100, Ninate 11, Georpon 71, Tetraonic 1357, Cremapor-el, Chemal la-9,Silwet L7900, Surfynly468, Surfactant 10G, and Tween 80 might also beused but they did not provide good results with the preferred elutionsolution, the CCM 1-hexanal and mPDA.

In the absence of SDS the polymerization and aldehyde response asdiscussed below is severely inhibited. mPDA is highly water soluble andthe presence of SDS may provide a scaffold for organizing andorientating mPDA into a matrix to facilitate the polymerizationreaction.

The solvent can include an aqueous solution of EtOH, MeOH, propanol, andisopropanol, with 15% EtOH preferred.

The molar ratio of salt concentration to phenylene diamine concentrationis important. Generally the ratio should range from 0.03 to 0.5. For theCCM 1-hexanal, a molar ratio of mPDA to NaCl of 0.165 was found toprovide optimal response.

The temperature for practicing the method of the present inventionpreferably ranges from 15 to 35° C., with 25 to 30° C. more preferred.

For the aldehydes such as 1-hexanal, one preferred embodiment of theelution solution comprises 33 mM NaCl, 50 mM Citrate, pH 2.5, 15% EtOH,and 0.2% SDS. Other preferred elution solutions include 50 mM Citrate,pH2.5, 15% propanol and 0.4% sodium decyl sulfate.

Using the elution solution containing a phenylene diamine derivative,the CCM is eluted into the phenylene diamine solution to form afluorescing solution. The CCM and the mPDA react to form a fluorogenicspecies, the presence of which in the fluorescing solution is detectedby measuring the fluorescence emitted by the fluorogenic species in thefluorescing solution.

The aldehyde content is quantitated by monitoring the signal rise(end-point) and/or rate of signal change (kinetic) which varies as afunction of aldehyde concentration for a given mPDA concentration, andcomparing such data with a carbonyl population sample of the breath. Inpractice the impact of carbonyls other than the selected carbonyl mustbe filtered out. There are two general assay format or detection modes.They are generically described as end-point and kinetic. In an end-pointassay the system is incubated for a set time and the signal read. Thesignal at that point reflects the amount of analyte in the system. For apositive assay, the greater the concentration of the analyte, thegreater the signal increase. In a kinetic assay the rate of change ismonitored for a set duration. The rate of change is correlated to theamount of analyte. Preferably the end-point assay is employed with thepresent invention.

Assay measurements can be made on a typical fluorescence spectrometerincluding conventional scanning spectrometer, plate-reader or LED/diodebased spectrometer following standard assay practices. To illustrate,the data displayed in FIG. 31 was acquired by mixing a total of 2 mL ofthe reaction solution and aldehyde into a standard fluorescence cuvetteand measuring the intensity increase using an LED/diode spectrometer atparticular time slices to simulate an end-point determination. TheLED/diode spectrometer utilized consisted of an Ocean Optics Jazzspectrometer with LED source and diode detection coupled via fiberoptics to a Qpod-e (Quantum Northwest) temperature controlledfluorescence sample holder. The 405 nm excitation was produced with aviolet LED (volts: 3.3 V, I: 0.03 A). The signal was detected using aILX-5118 diode detection with emission set at 495-505 nm band pass and250 msec integration. Like most fluorescence based assays, optimalsettings are dependent upon the throughput and stray light rejectioncharacteristics of the spectrometer used and must be empiricallydetermined for each instrument.

In one preferred embodiment, the phenylene diamine derivative reactswith the CCM in solution to produce a fluorescence emitting orfluorogenic species. It is believed that the phenylene diaminederivative oxidatively couples to the CCM and the phenylene diaminederivative polymerizes to dimers, trimers, oligomers and/or polymers. Itis not clear if the CCM actually becomes part of the growing polymer,although the polymerization is modulated by the presence of CCM andthere is a dose response.

The process of using a CCM to polymerize the phenylene diaminederivative may be described as dispersion polymerization. Poly-phenylenediamines have been used to construct nanostructures and colloidaldispersions of different shapes, tubes, spheres and the like. However,if the polymerization results in large high molecular weight structuresthen precipitation occurs in the solution, which, in the presentinvention, may handicap optical detection. Thus the ingredients used inthe method of the present invention must be chosen to avoid havingelements in the fluorescing solution that inhibit detection andquantitation of the CCM.

The present invention utilizes the ability of CCM to modulate (initiate,catalyze and accelerate) the oxidative coupling and polymerization ofphenylene diamine derivatives to detect and quantitate trace aldehydes,ketones and carbonyl containing analytes in a biological sample.Oxidative coupling and polymerization of phenylene diamine generateschromophoric and fluorogenic species. In the case of mPDA and aldehydes,the formation of polymers or multimers gives rise to a broad opticalabsorbance band at 405 nm and an associated emission band at 505 nm. Themonomer absorbance is found in the UV region <350 nm. As a result theproduction of the polymer can be conveniently followed by eitherconventional absorbance or fluorescence spectroscopy. In this regard, itshould be appreciated that the absorbance and emission bands may varydepending upon the CCM and phenylene diamine derivative chosen, but allsuch bands useful in practicing this invention are part of theinvention.

For example, with reference to FIG. 31, the emission spectrum of thereaction of mPDA in the presence of 1 μM 1-hexanal as a function of timeis shown. The conditions of the fluorescing solution are: 1 μM1-hexanal, 5.4 mM mPDA, 33 mM NaCl, 50 mM citrate (pH 2.5), 15% EtOH,and 0.1% SDS. The emission increases dramatically as a function of time.

With reference to FIG. 32, the reaction and responses with and withoutaldehyde (“blank”) are observed. The conditions of the fluorescingsolution are: 1 μM 1-hexanal, 5.4 mM mPDA, 33 mM NaCl, 50 mM citrate (pH2.5), 15% EtOH, and 0.1% SDS. The extent of the emission increase andthe rate of increase are dependent upon the concentration of aldehyde inthe phenylene diamine solution. At greater aldehyde concentrations, alarger and more rapid signal increase is observed. In the absence ofaldehyde, the “blank” under goes a slow gradual small signal riseindicative of the slow polymerization of mPDA under the conditionsexamined. The polymerization is presumably due to the presence of traceoxidants such as iron, reactive oxygen species and other initiators.With the addition of a CCM, a significant signal enhancement over theblank or background is observed. Of particular note is that the rate ofchange is easily followed. As a result the detection system is amenableto both kinetic and end-point assay designs and detection modalities.The response can be quantitated at specific time points, e.g., 15minutes (time slice) or by monitoring the slope as a function ofaldehyde. The kinetic rate is slow enough that rapid and high precisionof reactant additions is not required. The modulation of thepolymerization reaction by a CCM such as an aldehyde and its use as aCCM quantitative sensor is another novel discovery and applicationdescribed in this specification. Other alternatives including labeling,painting or tagging the CCM for subsequent analysis.

With reference to FIGS. 33A, 33B and 33C, the CCM induced polymerizationreaction with the phenylene diamine derivative is shown to be sensitiveto environmental conditions, and components of the reaction system suchas the concentration of SDS. The conditions of the fluorescing solutionin these figures are: 1 μM 1-hexanal, 5.4 mM mPDA, 33 mM NaCl, 50 mMcitrate (pH 2.5), and 15% EtOH. For example, the reaction and aldehydeassay performance is dependent upon salt content, mPDA content,surfactant, pH and temperature. Since the reaction involves a“quasi-phase” transition from monomer to polymer insufficient mPDAconcentration yields a slow reaction with limited signal change. Incontrast, a large excess of mPDA results in a very rapid reaction andthe formation of insoluble precipitates that limit optical detection. Inaddition, a large excess results in increased background or “blank”signal.

With reference to FIG. 33A, the signal increases as function of SDSconcentration. At an SDS concentration of 0.4%, the signal increase isalmost 3 times the signal observed at 0.2%.

FIGS. 33B and 33C show a comparison of the aldehyde response versus theblank for 0.2% SDS and 0.4% SDS, respectively. The increase in SDSconcentration also results in an increase in “blank” or backgroundsignal. Both the signal and background are modulated by SDSconcentration and the optimized SDS concentration cannot be determinedby monitoring the signal response alone. As a result the SDSconcentration must be optimized to provide the greatest discriminationbetween signal and background signal generation. For the embodimentspecified, the optimal SDS concentration falls within a narrowconcentration band, and small deviations can result in increasedvariability and limit the assay sensitivity.

With reference to FIG. 34, the fluorescence response for mPDA as afunction of 1-hexanal concentration is displayed, with the backgroundcorrected. A linear response is observed form 0.1 to 1 μM 1-hexanal. Thedata points are the average of triplicate samples. The signal ismeasured at 20 minutes after the aldehyde is added to the phenylenediamine solution. Under these conditions, 10.8 mM mPDA, 65.5 NaCl, 50 mMcitrate (pH 2.5), 0.2% SDS at 25° C., a solution limit of detection(LOD) of 0.1 μM can be achieved.

With reference to the chart in FIG. 35, mPDA exhibits a differentialresponse for aliphatic aldehydes as a function of chain length. Thechart reflects the fluorescence signal at 20 minutes after aldehydeaddition, and the following conditions: 5.4 mM mPDA, 33 mM NaCl, 50 mMcitrate (pH 2.5), 15% EtOH, and 0.1% SDS. The signal is measured at 20minutes and this time-slice serves as pseudo end-point analysis method.For aliphatic aldehydes the relative response increases with aliphaticchain length. The response of acetylaldehyde is only 12% of the responseobserved for 1-hexanal. In contrast, the response of decyl (C₁₀)aldehyde is 30% greater than for 1-hexanal.

The nature of the aromatic diamine is also important to consider inemploying the method of the present invention. O-PDA is highly reactiveand undergoes rapid general oxidation. The high reactivity of o-PDAprecludes its use as an aldehyde sensor in the preferred embodiment ofthe present invention. With reference to FIG. 36, the relativefluorescence response of a subset of diamines is displayed andillustrates the influence of both position and electronic effects on thealdehyde fluorescence response. Traditional aromatic electron donatingand withdrawing effects should modulate the reactivity andsusceptibility of the phenylene diamine derivative towardpolymerization. An aldehyde response was not observed for bothnitrophenylenediamine and naphthalenediamine under the preferredconditions, even when exposed to excess aldehyde. It has been found thataldehyde detection is based on the modulation of the polymerization ofthe reaction. If the molecule chosen is highly reactive and easilyinduced to polymerization then general oxidants can stimulate thereaction process and may limit its utility as a sensor. On the otherhand, if the molecule is “too” stabilized, the polymerization processbecomes inhibited and cannot be adequately stimulated by aldehyde andwill require a much stronger oxidant to yield a response.

The present invention discussed above also includes a device foremploying the method of the present invention. The device comprises abreath chamber preferably made of plastic and having a substrate in thebreath chamber. The substrate is made from the materials discussed aboveand preferably silica. The substrate supports a carbonyl containingmoiety from an animal's breath, e.g. aldehydes. The device also includesa fluid chamber. The fluid chamber includes an aqueous solutioncomprising an alcohol (e.g., 15% EtOH), a salt (e.g., NaCl), asurfactant (e.g., SDS), and a buffer (e.g. citrate). The solution canalso comprise a phenylene diamine derivative such as mPDA.

The following example demonstrates one way to use the present inventionto determine whether the sample breath of a human contains measurablealdehyde concentration and the concentration of the aldehyde in thebreath. Employing the methodology discussed above, a series offluorescence measurements are preformed to provide standards for variousspecific aldehydes and mixtures thereof that are known to be containedin a human breath sample (a population), and standards forconcentrations of such various standards and mixtures thereof. Usingthese standards, the presence in a sample of human breath of aparticular aldehyde or mixture of aldehydes and the concentration ofsuch particular aldehyde or mixture of aldehydes can be determined. Ingeneral in one embodiment, the steps are as follows:

-   -   a. Capturing the aldehydes from the human breath sample on        silica;    -   b. Forming a solution comprising a salt, a buffer, a surfactant        in an alcohol in mildly acidic conditions;    -   c. Adding a phenylene diamine derivative to the solution of step        b;    -   d. Eluting the captured aldehydes into the solution of step c;    -   e. Determining the fluorescence signal of the solution of step        c;    -   f. Determining the fluorescence signal of the solution of step        d;    -   g. Subtract the fluorescence signal from step e from the        fluorescence signal from step f; and    -   h. Comparing the net resulting fluorescence signal from step g        with standard fluorescence of known aldehydes (a calibration        curve, i.e., a response to known concentrations via an assay) to        determine the concentration of aldehydes in the fluorescing        solution. Simply put, this is a comparison of “y” axis values to        provide the “x” axis value, or alternatively, solve of x knowing        y and the calibration function y=f(x).

In another embodiment of the present invention, the substrate can bepre-loaded with an active reactive capture agent which covalentlyattaches to the CCM (the “Agent”) including without limitation afluorescent hydrazine or aminooxy compound. Some examples of aminooxycompounds are as follows: aminooxy 5(6) tetramethylrhodamine (aminooxy5(6) TAMRA), with a single isomer of either 5 or 6 preferred; andaminooxy 5(6) carboxyfluorescein (aminooxy 5(6) FAM), with a singleisomer of either 5 or 6 preferred, for example aminooxy-05-5-FAM. Othersinclude aminooxy 7-amino-3-acetyl-4 methylcourman-6-sulfonic acid;5-aminoxy acetic acid rhodamine B; and dinitrophenylhydrazin. In theforegoing examples, the reactive group is specified without the linkagegroup, which would be well known to those of skill in the art. Inaddition to the foregoing, the hydrazine or hydrazide versions areincluded within the present invention. Preferably the Agent is somewhatpolar.

For example, for a substrate consisting of 200 mg of 50-270 mesh (300-50μm) particle with a bed diameter of 12.5 mm, the amount of the Agent canbe from 5.5 mg to 0.1 mg, and preferably from 2.5 mg to 0.4 mg.

In yet another embodiment of the present invention, a two-solutionmethodology is used. After the substrate is loaded with the CCM, the CCMis eluted into a first elution solution or “rinse” solution comprisinggenerally 30% ethanol and preferably 50 mM citrate, 30% ethanol at ph2.5. The Agent is added to the rinse solution thereby resulting inpainted CCM. This solution is then passed through another substrate,preferably a silica frit stack, to capture the painted CCM. The paintedCCM is then eluted from the substrate with the painted CCM capturedtherein using a second elution solution or “rinse” solution comprisinggreater than 50% acetonitrile and preferably 90% ethanol. One of thebenefits of this second embodiment is that a baseline reading is notnecessary to remove noise.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise,” “comprising,” and thelike are to be construed in an inclusive sense, as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to.” As used herein, the terms “connected,”“coupled,” or any variant thereof, means any connection or coupling,either direct or indirect, between two or more elements; the coupling ofconnection between the elements can be physical, logical, or acombination thereof. Additionally, the words “herein,” “above,” “below,”and words of similar import, when used in this application, shall referto this application as a whole and not to any particular portions ofthis application. Where the context permits, words in the above DetailedDescription of the Preferred Embodiments using the singular or pluralnumber may also include the plural or singular number respectively. Theword “or” in reference to a list of two or more items, covers all of thefollowing interpretations of the word: any of the items in the list, allof the items in the list, and any combination of the items in the list.

The above-detailed description of embodiments of the disclosure is notintended to be exhaustive or to limit the teachings to the precise formdisclosed above. While specific embodiments of and examples for thedisclosure are described above for illustrative purposes, variousequivalent modifications are possible within the scope of thedisclosure, as those skilled in the relevant art will recognize. Furtherany specific numbers noted herein are only examples: alternativeimplementations may employ differing values or ranges.

The above-detailed description of embodiments of the disclosure is notintended to be exhaustive or to limit the teachings to the precise formdisclosed above. While specific embodiments of and examples for thedisclosure are described above for illustrative purposes, variousequivalent modifications are possible within the scope of thedisclosure, as those skilled in the relevant art will recognize.Further, any specific numbers noted herein are only examples:alternative implementations may employ differing values, measurements orranges. It will be appreciated that any dimensions given herein are onlyexemplary and that none of the dimensions or descriptions are limitingon the present invention.

The teachings of the disclosure provided herein can be applied to othersystems, not necessarily the system described above. The elements andacts of the various embodiments described above can be combined toprovide further embodiments.

Any patents and applications and other references noted above, includingany that may be listed in accompanying filing papers, are incorporatedherein by reference in their entirety. Aspects of the disclosure can bemodified, if necessary, to employ the systems, functions, and conceptsof the various references described above to provide yet furtherembodiments of the disclosure.

These and other changes can be made to the disclosure in light of theabove Detailed Description of the Preferred Embodiments. While the abovedescription describes certain embodiments of the disclosure, anddescribes the best mode contemplated, no matter how detailed the aboveappears in text, the teachings can be practiced in many ways. Details ofthe system may vary considerably in its implementation details, whilestill being encompassed by the subject matter disclosed herein. As notedabove, particular terminology used when describing certain features oraspects of the disclosure should not be taken to imply that theterminology is being redefined herein to be restricted to any specificcharacteristics, features or aspects of the disclosure with which thatterminology is associated. In general, the terms used in the followingclaims should not be construed to limit the disclosures to the specificembodiments disclosed in the specification unless the above DetailedDescription of the Preferred Embodiments section explicitly defines suchterms. Accordingly, the actual scope of the disclosure encompasses notonly the disclosed embodiments, but also all equivalent ways ofpracticing or implementing the disclosure under the claims.

Accordingly, although exemplary embodiments of the invention have beenshown and described, it is to be understood that all the terms usedherein are descriptive rather than limiting, and that many changes,modifications, and substitutions may be made by one having ordinaryskill in the art without departing from the spirit and scope of theinvention.

What is claimed is:
 1. A breath capture assembly comprising: a handleassembly that includes an elongated main body portion that defines ahandle interior, a cap disposed at an end of the main body portion thatincludes a pressure opening defined therein, and a pressure transducerdisposed in the handle interior, and an analysis cartridge received onan upper end of the handle assembly, wherein the analysis cartridgeincludes a main body portion that includes an upper portion that definesa breath chamber, and a lower portion that defines a fluid chamber,wherein the breath chamber includes a breath entry opening, a breathexit opening and a breath path therebetween, and a filter assembly thatis movable along a filter assembly path between a first position and asecond position, wherein the filter assembly has an opening definedtherethrough, and wherein in the first position, the opening partiallydefines the breath chamber and is part of the breath path and in thesecond position the opening partially defines the fluid chamber.
 2. Thebreath capture assembly of claim 1 wherein a pressure measurement holeis defined in a wall of the upper portion of the analysis cartridge,wherein the pressure measurement hole communicates the breath chamberwith a pressure tunnel that extends through the main body portion, andwherein a pressure path is defined from the breath chamber, through thepressure measurement hole, the pressure tunnel, the pressure opening andto the pressure transducer.
 3. The breath capture assembly of claim 2wherein the cap of the handle assembly includes a pressure protrusionextending upwardly therefrom that is sealingly received in a pressurerecess in the analysis cartridge, wherein the pressure recess is incommunication with the pressure tunnel, and wherein the pressure openingis defined in the pressure protrusion.
 4. The breath capture assembly ofclaim 2 wherein a hollow extension extends downwardly from the cap ofthe handle assembly and into the handle interior, and wherein the hollowextension is part of the pressure path.
 5. The breath capture assemblyof claim 4 further comprising a pressure tube that is received on thehollow extension and is in the pressure path between the hollowextension and the pressure transducer.
 6. The breath capture assembly ofclaim 1 wherein the cap includes a seat defined therearound, and whereina collar depending downwardly from the analysis cartridge is received onthe seat.
 7. The breath capture assembly of claim 6 wherein the capincludes an attachment protrusion extending radially outwardlytherefrom, and wherein the attachment protrusion is received in anattachment recess defined in the collar of the analysis cartridge. 8.The breath capture assembly of claim 1 wherein the breath chamber issealed from the fluid chamber.
 9. The breath capture assembly of claim 8wherein the analysis cartridge includes a breakable barrier disposedbetween the breath chamber and the fluid chamber when the filterassembly is in the first position to seal the breath chamber from thefluid chamber.
 10. The breath capture assembly of claim 1 wherein thefilter assembly includes first and second filters positioned in theopening, wherein the first and second filters define a substrate spacetherebetween, and wherein a carbonyl containing moiety capture materialis disposed in the substrate space.
 11. The breath capture assembly ofclaim 10 wherein the carbonyl containing moiety capture material issilica.
 12. The breath capture assembly of claim 1 wherein the analysiscartridge includes a phenylene diamine derivative disposed therein. 13.The breath capture assembly of claim 12 further comprising an ampulemember having a fluorescence chromophore space with the phenylenediamine derivative disposed therein, wherein the fluid chamber includesan elution solution disposed therein, wherein the ampule member ismovable between a first position where the phenylene diamine derivativeis separated from the elution solution and a second position where thephenylene diamine derivative is disposed in the elution solution. 14.The breath capture assembly of claim 13 wherein the phenylene diaminederivative is m-phenylene diamine.
 15. The breath capture assembly ofclaim 12 wherein the phenylene diamine derivative is m-phenylenediamine.